Thermo Scientific Pierce Protein Purification Technical Handbook

Agarose

Bead

NH2

H2N

Thermo Scientiﬁc Pierce Protein

Puriﬁcation Technical Handbook

Version 2

Introduction for Protein Puriﬁcation 1-5

Puriﬁcation Accessories – Protein Extraction,

Binding and Elution Buffers 6-11

Protease and Phosphatase Inhibitors 8

for Protein Puriﬁcation

Buffers for Protein Puriﬁcation 9

Spin Cups and Columns 10

Disposable Plastic and Centrifuge Columns 11

Fusion Protein Puriﬁcation 12-21

His-Tagged Protein Puriﬁcation Resin 14-15

Cobalt Resin, Spin Columns and 16-17

Chromatography Cartridges

High-quality puriﬁcation of GST-fusion proteins 18-19

GST- and PolyHis-Tagged Pull-Down Assay Kits 20-21

Covalent Coupling of Afﬁnity Ligands

to Chromatography Supports 22-35

Covalent Immobilization of Ligands 22-25

Products for Immobilizing Ligands 26-29

through Primary Amines

Products for Immobilizing Ligands 30-32

through Sulfhydryl Groups

Products for Immobilizing Ligands 33

through Carbonyl Groups

Products for Immobilizing Ligands 34-35

through Carboxyl Groups

IP/Co-IP 36-45

Immunoprecipitation 36

Traditional Methods vs. 37

Thermo Scientiﬁc Pierce Innovations for Co-IP

Approaches to Co-IP Free of Antibody Interference 37

Optimization Parameters in IP and Co-IP 38

Evaluating a Co-IP-Captured Interaction 38

IP and Co-IP Kits 39-45

Protein Enrichment 46-57

Phosphoprotein Enrichment Kits 46-47

SH2 Domain Phosphotyrosine Capture Kits 48-50

Use of Titanium Dioxide for 51-52

Phosphopeptide Enrichment

Pierce Fe-NTA Phosphopeptide Enrichment Kit 53-54

Cell Surface Protein Isolation Kit 55

Glycoprotein Isolation Kits 56

Ubiquitin Enrichment Kit 57

Antibody Puriﬁcation 58-67

Overview 58

Immobilized Protein L, Protein A, Protein G 59-61

and Protein A/G

IgG Binding and Elution Buffers for 62

Protein A, G, A/G and L

Melon Gel Puriﬁcation Products 63

Thiophilic Gel Antibody Puriﬁcation 64

IgM and IgA Puriﬁcation 65

Avidin:Biotin Binding 66-69

Biotin-binding Proteins 66

Immobilized Avidin Products 67

Immobilized Streptavidin Products 67

Immobilized NeutrAvidin Products 68

Immobilized Monomeric Avidin and Kit 68

Immobilized Iminobiotin and Biotin 69

FPLC Cartridges 70-77

Overview 70

His-tagged Protein FPLC Puriﬁcation 71-72

GST-Tagged Protein FPLC Puriﬁcation 72

Antibody FPLC Puriﬁcation 73-75

Phosphoprotein FPLC Puriﬁcation 75

Biotinylated Protein FPLC Puriﬁcation 76

Protein Desalting 77

Afﬁnity Supports 78-80

Table of Contents

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch ofﬁce or distributor.

General Protein Puriﬁcation Techniques

Protein puriﬁcation is essential for a host of biochemical appli-

cations. However, with thousands of proteins each displaying

unique characteristics, it is important to develop a strategy for

puriﬁcation that delivers the correct yield, purity and activity

needed for downstream applications. For low resolution/high yield

protein puriﬁcation, methods such as fractional precipitation using

salts such as ammonium sulfate exist. For applications requiring

the highest purity and relatively small amounts of protein, afﬁnity

puriﬁcation techniques can be chosen to selectively extract a

target protein from the complex mixture of proteins found in cell

or tissue extracts. Table 1 summarizes a few general strategies.

Each of these protein puriﬁcation techniques requires speciﬁc

buffers (mobile phase), chromatography resins (solid phase) and

column accessories. These three components can be used in a

variety of different conﬁgurations, based on the scale of puriﬁca-

tion and available equipment. Common formats include:

Batch puriﬁcation

Mixing the mobile and stationary phases in a conical tube and

separating the two via centrifugation. Batch method puriﬁcation

can be performed at any scale. However, it is most commonly

reserved for microcentrifuge tube scale puriﬁcations involving

10-200μL of resin. In batch method puriﬁcation, wash and elution

fractions are separated from the resin after centrifuging to pellet

the resin beads. The liquid cannot be removed completely because

some of it is contained within the volume of porous bead pellet.

Consequently, a portion of each fraction about equal to the volume

of resin used is left behind in the pellet, making washes and elution

somewhat inefﬁcient.

Gravity ﬂow chromatography

Passively adding the mobile phase to

packed columns, without mechanically

increasing the ﬂow rate. Gravity ﬂow com-

monly uses 1mL- to 5mL-packed columns

set-up on a bench or in a chromatography

refrigerator. Larger columns can be packed

to support larger scale protein puriﬁcation. However, the weight of

the resin in the column must be considered to prevent damage to

resin at the bottom of the column. Gravity ﬂow allows for extended

binding, washing and elution times, which is ideal for samples with

low binding afﬁnity.

Introduction to Protein Puriﬁcation

Table 1. Summary of protein puriﬁcation techniques.

Resolution/ purity Technique Protein Yield Description

Low

Ammonium sulfate precipitation High Fractional precipitation of proteins based on their solubility in salt solutions of varying saturation

Hydrophobic interaction chromatography High Separation of proteins based in their surface hydrophobicity

Size exclusion chromatography (SEC) Medium Separation of proteins based on molecular size

Medium

Ion exchange chromatography Medium Separation based on protein charge at a particular pH

Gel electrophoresis Medium Two-step separation of protein based on size using polyacrylimide gel electrophoresis

(SDS-PAGE) and charge using isoelectric focusing. Note: This is a denaturing method that results

in a complete loss of protein activity.

Proteome fractionation Medium Enrichment of classes of proteins, such as:

• phosphoproteins using immobilized metal afﬁnity chromatography (IMAC)

• glycoproteins using immobilized lectins

• nitrosylated or palmitylated proteins using the biotin switch assay

• organelle-speciﬁc proteomes using cellular fractionation techniques

High

Afﬁnity puriﬁcation Low Selective puriﬁcation using afﬁnity tags attached to the target gene, such as polyhistidine (6xHis),

glutathione S-transferase (GST) or maltose binding protein (MBP)

Immunoprecipitation Lowest Antibody-based extraction of a single protein species, typically from cell or tissue lysatesh

For more information, or to download product instructions, visit www.thermoscientiﬁc.com/pierce

Spin cup puriﬁcation

Separating the mobile and stationary

phases using centrifugation in packed spin

tubes with ﬁlters that retain resin in column.

Spin puriﬁcation can also be performed

with spin plates, where each well of a

96-well microtiter plate has a ﬁlter base

and is packed with the appropriate chromotagraphy resin. The

spin cup puriﬁcation method provides improved efﬁciency of wash

and elution steps compared to the batch method. Centrifugation

separates the liquid fraction by pulling it thoroughly from the resin,

which is retained within the spin cup apparatus. Spin cup puriﬁca-

tion is most appropriate when 50-300μL of immobilized ligand resin

is used.

Magnetic puriﬁcation

Isolating the stationary phase using mag-

nets. Magnetic beads are commonly iron

oxide particles which are coated with a

ligand to purify a protein target. An example

of this is a magnetic particle coated with

reduced glutathione (GSH) used to purify

GST-tagged recombinant proteins (Product # 88821). Advantages of

using magnetic beads include easy handling, minimal loss of resin

from pipetting and compatibility with high throughout automated

systems such as the Thermo Scientiﬁc KingFisher 96 instrument.

Fast protein liquid chromotagraphy (FPLC)

Using chromatography cartridges pre-

packed with the stationary phase and

a series of pumps and UV detectors to

move and monitor the mobile phase. The

advantages of using FPLC include reduced

puriﬁcation times and the ability to improve

resolution by linking multiple columns in

tandem for greater separation.

Afﬁnity Puriﬁcation

Various methods are used to enrich or purify a target protein from

other proteins and components in a crude cell lysate or other

sample. The most powerful of these methods is afﬁnity puriﬁcation,

also called afﬁnity chromatography, whereby the protein of

interest is puriﬁed using its speciﬁc binding properties to an

immobilized ligand.

Afﬁnity puriﬁcation makes use of speciﬁc binding that occurs

between molecules and is used extensively for the isolation of

biological molecules. A single pass through an afﬁnity column

can achieve a 1,000- to 10,000-fold puriﬁcation of a ligand from a

crude mixture. From a single afﬁnity puriﬁcation step, it is possible

to isolate a compound in a form pure enough to obtain a single

band upon SDS-PAGE analysis. We offer a number of immobilized

protein or ligand products for afﬁnity puriﬁcation of antibodies,

fusion-tagged proteins, biotinylated proteins and other proteins

for which an afﬁnity ligand is available.

In afﬁnity puriﬁcation, a ligand is immobilized to a solid support.

Once immobilized, it speciﬁcally binds its partner under mild buffer

conditions (often physiological conditions such as phosphate

buffered saline). After binding to the partner molecule, the support

is washed with additional buffer to remove unbound components

of the sample. An elution buffer is added, disrupting the interaction

between the ligand and its binding partner by pH extremes

(low or high), high salt, detergents, chaotrophic agents or the

removal of some factor required for the pair to bind. Once

released, the binding partner can be recovered from the support

using additional elution buffer. The elution buffer can then be

exchanged by dialysis or desalting into a more suitable buffer

for storage or downstream analysis.

Activated afﬁnity support products and kits enable a researcher to

immobilize nearly any type of ligand to purify its binding partner(s).

For example, if a peptide antigen is used to immunize animals and

produce antibodies, the same peptide can be immobilized to a

gel support and used to afﬁnity-purify the speciﬁc antibody from

animal serum. Alternatively, if a speciﬁc antibody is available

against a particular protein of interest, it can be immobilized to

a support and used to afﬁnity-purify the protein from crude cell

lysate. Puriﬁcation with respect to nearly any binding interaction

can be made by this approach.

Introduction to Protein Puriﬁcation

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch ofﬁce or distributor.

Afﬁnity puriﬁcation products using either immobilized ligands

or activated afﬁnity support chemistries are available for use

in several different formats. Most commonly, porous beaded gel

supports are used for gravity-column, spin-column or batch-scale

puriﬁcation procedures. Coated microplates are available for

high-throughput screening applications, and magnetic particles

are especially useful for automated protein puriﬁcation.

Proteins and other macromolecules of interest can be puriﬁed

from crude extracts or other complex mixtures using a variety

of methods. Precipitation is perhaps the simplest method for

separating one type of macromolecule from another. For example,

nucleic acids can be precipitated and thereby puriﬁed from

undesired molecules in solution using ethanol, and proteins can

be selectively precipitated in the presence of ammonium sulfate.

Most puriﬁcation methods involve some form of chromatography

whereby molecules in solution (mobile phase) are separated based

on differences in chemical or physical interaction with a station-

ary material (solid phase). Gel ﬁltration (also called desalting, size

exclusion chromatography or SEC) uses a porous gel material to

separate molecules based on size; large molecules are excluded

from the internal spaces of the gel material while small molecules

enter the resin pores, resulting in a longer path through the col-

umn. In ion exchange chromatography, molecules are separated

according to the strength of their overall ionic interaction with a

solid-phase material. By manipulating buffer conditions, molecules

of greater or lesser ionic character can be bound to or dissociated

from the solid-phase material.

In contrast, afﬁnity chromatography or afﬁnity puriﬁcation makes

use of speciﬁc binding interactions between molecules. A particular

ligand is chemically immobilized or “coupled” to a solid support

so that when a complex mixture is passed over the column, only

those molecules having speciﬁc binding afﬁnity to the ligand are

puriﬁed. Afﬁnity puriﬁcation generally involves the following steps:

1. Incubate crude sample with the immobilized ligand support

material to allow the target molecule in the sample to bind to

the immobilized ligand.

2. Wash away unbound sample components from solid support.

3. Elute (dissociate and recover) the target molecule from the

immobilized ligand by altering the buffer conditions so that

the binding interaction no longer occurs.

Ligands that bind to general classes of proteins (e.g., Protein A for

antibodies) or commonly used fusion protein tags (e.g., glutathione

for GST-tagged proteins) are available in pre-immobilized forms

ready to use for afﬁnity puriﬁcation. Alternatively, more specialized

ligands such as speciﬁc antibodies or antigens of interest can be

immobilized using one of several activated afﬁnity supports; for

example, a peptide antigen can be immobilized to a support and

used to purify antibodies that recognize the peptide.

Most commonly, ligands are immobilized or “coupled” directly to

solid support material by formation of covalent chemical bonds

between particular functional groups on the ligand (e.g., primary

amines, sulfhydryls, carboxylic acids, aldehydes) and reactive

groups on the support. However, other coupling approaches are

also possible. In the Thermo Scientiﬁc GST Orientation Kit

(Product # 78201), for example, a GST-tagged fusion protein is ﬁrst

bound to an immobilized glutathione support by afﬁnity interaction

with the GST tag and then chemically crosslinked to the support.

The immobilized GST-tagged fusion protein can then be used to

afﬁnity-purify its binding partner(s). Likewise, the Thermo Scientiﬁc

Pierce Crosslink Immunoprecipitation Kits (Product # 26147) and

Thermo Scientiﬁc IgG Orientation Kits (Product # 44990) involve

binding and subsequent crosslinking of an antibody to immobilized

Protein A, A/G.

Historically, researchers have used afﬁnity puriﬁcation primarily

to purify individual molecules of interest. Increasingly, proteomics

research focuses on determination of disease states, cell

differentiation, normal physiological functions and drug discovery

involving interaction and expression of multiple molecules rather

than individual targets. Consequently, the use of afﬁnity methods

has expanded to puriﬁcation of native molecular complexes and

forms the basis for co-immunoprecipitation (co-IP) and “pull-down”

assays involving protein:protein interactions.

1. Immobilize the antigen to

an appropriate support.

4. Bind anti-antigen

antibodies.

5. Wash off unbound

antibodies.

6. Elute anti-antigen

antibodies.

2. Quench the unreacted

sites and wash.

3. Add the antibody solution.

5ml

4ml

3ml

2ml

1ml

5ml

4ml

3ml

2ml

1ml

5ml

4ml

3ml

2ml

1ml

5ml

4ml

3ml

2ml

1ml

5ml

4ml

5ml

4ml

5ml

4ml

Antigen

TBS

Typical antibody puriﬁcation using an immobilized antigen column.

For more information, or to download product instructions, visit www.thermoscientiﬁc.com/pierce

Afﬁnity Puriﬁcation Supports

Afﬁnity puriﬁcation involves the separation of molecules in solution

(mobile phase) based on differences in binding interaction with a

ligand that is immobilized to a stationary material (solid phase). The

solid phase in afﬁnity puriﬁcation is a support or matrix material

that a biospeciﬁc ligand may be covalently attached. Typically, the

material to be used as an afﬁnity matrix is insoluble in the system

in which the target molecule is found. Usually, but not always,

the insoluble matrix is a solid. Hundreds of substances have been

described and employed as afﬁnity matrices.

Useful afﬁnity supports are those with a high surface area to

volume ratio, chemical groups that are easily modiﬁed for covalent

attachment of ligands, minimal nonspeciﬁc binding properties,

good ﬂow characteristics, and mechanical and chemical stability.

When choosing an afﬁnity support or matrix for any separation,

the most important question to answer is whether a reliable

commercial source exists for the desired matrix material in the

quantities required. Fortunately, we offer a wide range of practical

and efﬁcient matrices in volumes ranging from 1mL to much larger

bulk quantities.

Porous Beaded Resins

Porous beaded supports generally provide the most useful proper-

ties for afﬁnity puriﬁcation of proteins. We offer afﬁnity puriﬁca-

tion products in two main porous gel support formats: crosslinked

beaded agarose and Thermo Scientiﬁc UltraLink Biosupport.

The various features of these two supports are listed in the

accompanying table. Agarose is good for routine applications but

crushes easily, making it suitable for gravity-ﬂow column or small-

scale batch procedures using low-speed centrifugation. UltraLink

Biosupport is incompressible and can be used in high-pressure

applications with a peristaltic pump or other liquid chromatogra-

phy system. In addition, UltraLink

Supports display extremely low

nonspeciﬁc binding characteristics because they are polyacryl-

amide-based. Both supports perform well in typical gravity-ﬂow

and spin column puriﬁcation and immunoprecipitation procedures.

Magnetic Particles

When a matrix is required for afﬁnity puriﬁcation of cells within a

population, we recommend Thermo Scientiﬁc MagnaBind Beads.

Magnetic afﬁnity separation is a convenient method for isolating

antibodies, antigens, lectins, enzymes, nucleic acids and cells

while retaining biological activity. Samples containing the mole-

cule of interest are incubated with beads that are derivatized with

an antibody or other binding partner. A rare earth magnet is used

to pull the MagnaBind

™

Beads out of solution and onto a surface.

The buffer can be carefully removed, containing any non-bound

molecules or cells.

MagnaBind Beads consist of a silanized surface over an iron

oxide core (see Table 2). The silanized surface has been deriva-

tized to contain active groups, such as carboxylic acids or primary

amines, or speciﬁc afﬁnity molecules such as streptavidin; Protein

A; Protein G; or goat anti-mouse, anti-rabbit or anti-rat IgG. Due

to the nature of the MagnaBind Beads, strong elution conditions

are not recommended with these products. See page 80 for a

complete listing of MagnaBind Supports.

Introduction to Protein Puriﬁcation

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch ofﬁce or distributor.

Table 2. Characteristics of underivatized Thermo Scientiﬁc MagnaBind Beads.

Composition

Silanized iron oxide

Magnetization

25–35EMU/g

Type of Magnetization

Superparamagnetic (no magnetic memory)

Surface Area

>100m

Settling Rate

4% in 30 minutes

Effective Density

2.5g/mL

Number of Beads

1 x 10

beads/mg

pH Stability

Aqueous solution, above pH 4.0

Concentration

5mg/mL

Note: To establish a microbe-free preparation, MagnaBind Beads can be washed with

antibiotic medium or g-irradiated.

Microplates

Polystyrene microplates are another type of matrix commonly

used for immobilization of proteins. Proteins passively adsorb

to the polystyrene surface through hydrophobic interactions.

Generally, this adsorption of proteins onto the polystyrene surface

occurs best in carbonate/bicarbonate buffer at an alkaline pH

(9.0–9.5). In addition, polystyrene surfaces can be derivatized with

certain chemistries that will allow peptides and other nonprotein

molecules to adhere to the surface to perform afﬁnity assays in

the wells of the plates.

We offer precoated plates to allow researchers an easy-to-use,

consistent method for afﬁnity puriﬁcation or identiﬁcation of

speciﬁc molecules of interest. The plates offered include those

speciﬁc for fusion proteins (6xHis, GST and GFP), antibodies

(Protein A, Protein G, Protein A/G, Protein L, goat anti-mouse and

goat anti-rabbit IgG), biotin (streptavidin and Thermo Scientiﬁc

NeutrAvidin Protein) and those with reactive chemistries (maleic

anhydride and maleimide) to allow binding of nonprotein samples

that do not adsorb to the plastic microplate well surface. Only

selected microplate products are featured in this handbook. For

a complete selection of precoated plates, visit www.thermosci-

entific.com/pierce.

There are a variety of activated supports that allow a researcher

to purify proteins and other biological molecules of interest either

alone or when present in complexes with their binding partners.

Many of these supports are discussed on the following pages.

Physical properties of porous gel supports.

Support

4% Agarose

(crosslinked beaded agarose)

6% Agarose

(crosslinked beaded agarose)

Thermo Scientiﬁc UltraLink Biosupport

(co-polymer of crosslinked bis-acrylamide and azlactone)

Bead

45–165μm 45–165μm 50–80μm

Exclusion Limit

20,000,000 daltons 4,000,000 daltons 2,000,000 daltons (1,000 Å pore size)

Durability

Crushes under pressure Crushes under pressure Sturdy (>100 psi, 6.9 bar)*

Types of Chromatography

Gravity and small spin columns Gravity and small spin columns FPLC Systems, medium pressure, gravity ﬂow

Coupling Capacity

Medium Medium High

pH range

3–11 3–11 3–11

Form

Preswollen Preswollen Dry or Preswollen

* Note: The indicated maximum pressure of 100 psi refers to the maximum pressure

drop across the gel bed that the support can withstand. It does not necessarily

refer to the indicated system pressure shown on a liquid chromatography apparatus

because the system pressure may not actually be measuring the pressure drop

across the column. Typical system pressures are usually much higher due to

pumping through small I.D. tubing, auto-samplers, detectors, etc. When packed into

a 3mm ID x 14cm height glass column, these exclusive supports have been run to

approximately 650 psi (system pressure) with no visual compression of the gel or

adverse effects on chromatography. These columns can be run at linear ﬂow rates

or 85–3,000cm/hour with excellent separation characteristics.

For more information, or to download product instructions, visit www.thermoscientiﬁc.com/pierce

Protein Extraction, Binding and Elution Buffers

Protein puriﬁcation is preceeded by expression of the target

protein in a host organism. In in vitro protein expression, the

simple introduction of DNA and a bacteriophage RNA polymerase

to a cell free lysate initiates protein production, with no extrac-

tion reagents required before puriﬁcation. For in vivo protein

expression, such as in E. coli or tissue culture cells, expression

can be driven from constituitive promoters or it can be induced

chemically during bacterial growth or induced genetically

through transient DNA transfection into tissue culture cells.

For in vivo protein expression, the total protein content of a cell

culture or tissue sample must be extracted from the cell's mem-

branes and organelles before a chromatography resin can be

used for puriﬁcation. A list of Thermo Scientiﬁc Pierce Protein

Extraction Reagents for different cell and tissue type is listed

on page 7. Additionally, protease and phosphatase inhibitor

cocktails that can be used to preserve protein integrity during

extraction (see page 8).

Most afﬁnity puriﬁcation procedures involving protein:ligand inter-

actions use binding buffers, such as phosphate buffered saline

(PBS), at physiologic pH and ionic strength. This is especially true

when antibody:antigen or native protein:protein interactions are

the basis for the afﬁnity puriﬁcation. Once the binding interaction

occurs, the support is washed with additional buffer to remove

unbound components of the sample.

Nonspeciﬁc (e.g., simple ionic) binding interactions can be mini-

mized by moderate adjustments to salt concentration or by adding

low levels of detergent in the binding and/or wash buffer. Finally,

elution buffer is added to break the binding interaction and release

the target molecule, which is then collected in its puriﬁed form.

Elution buffer can dissociate binding partners by extremes of pH

(low or high), high salt (ionic strength), the use of detergents or

chaotropic agents that denature one or both of the molecules,

removal of a binding factor, or competition with a counter ligand.

In most cases, subsequent dialysis or desalting is required to

exchange the puriﬁed protein from elution buffer into a more

suitable buffer for storage or downstream analysis. For more

information on dialysis or desalting, download or request our

a free high-performance dialysis technical handbook.

The most widely used elution buffer for afﬁnity puriﬁcation of

proteins is 0.1M glycine•HCl, pH 2.5–3.0. This buffer effectively

dissociates most protein:protein and antibody:antigen binding

interactions without permanently affecting protein structure.

However, some antibodies and proteins are damaged by low pH,

so eluted protein fraction(s) should be neutralized immediately

by collecting the fractions in tubes containing 1/10th volume of

alkaline buffer such as 1M Tris•HCl, pH 8.5. Other elution buffers

for afﬁnity puriﬁcation of proteins are listed in the accompanying

table. In addition, we offer several preformulated binding and

elution buffers designed for afﬁnity puriﬁcation involving antibodies.

Common elution systems for protein afﬁnity puriﬁcation.

Condition Buffer

pH 100mm glycine•HCl, pH 2.5–3.0

100mm citric acid, pH 3.0

50–100mm triethylamine or triethanolamine, pH 11.5

150mm ammonium hydroxide, pH 10.5

Ionic strength and/or

chaotropic effects

3.5–4.0M magnesium chloride, pH 7.0 in 10mm Tris

5M lithium chloride in 10mm phosphate buffer, pH 7.2

2.5M sodium iodide, pH 7.5

0.2–3.0 sodium thiocyanate

Denaturing 2–6M guanidine•HCl

2–8M urea

1% deoxycholate

1 % SDS

Organic 10% dioxane

50% ethylene glycol, pH 8–11.5 (also chaotropic)

Competitor >0.1M counter ligand or analog

Protein Puriﬁcation Accessories

Cell Lysis Technical Handbook

This 50-page handbook provides

protocols and technical and product

information to help maximize results

for protein/gene expression studies.

The handbook provides helpful hints

and troubleshooting for cell lysis,

protein puriﬁcation, cell fractionation,

protease inhibitors and protein

refolding. (# 1601756)

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch ofﬁce or distributor.

Thermo Scientiﬁc Pierce Protein Extraction Reagents.

Name Description

Organisms/Samples

B-PER

- Bacterial Protein

Extraction Reagent

Product # 90084

Efﬁcient, gentle lysis and extraction of soluble proteins from E. coli and other

bacterial cells. Uses mild nonionic detergents to disrupt cells and solubilizing

proteins without denaturation, eliminating the need for harsh mechanical

procedures like sonication.

Gram(-) bacteria, S. aureus, H. pylori, E. coli strains

BL21(D3)>JM109>DH5a>M15, Archaebacteria,

nematodes and Acinetobacter sp.

B-PER II

Product # 78260

Similar to B-PER, but optimized for low cell density, or for proteins with low

expression levels.

Gram(-) bacteria, S. aureus, H. pylori, E. coli strains

BL21(D3)>JM109>DH5a>M15, Archaebacteria,

nematodes and Acinetobacter sp.

B-PER PBS

Product # 78266

Similar to B-PER, but in Phosphate Buffer. This amine free formulation is ideal

for amine-reactive labeling and/or crosslinking applications.

Gram(-) bacteria, S. aureus, H. pylori, E. coli strains

BL21(D3)>JM109>DH5a>M15, Archaebacteria,

nematodes and Acinetobacter sp.

B-PER with Enzymes

Product # 90078

Similar to B-PER, but kit contains DNAse I and lysozyme, which

improve cell membrane and DNA digestion for increased yields,

and increases the recovery of large molecular weight proteins

and insoluble proteins from inclusion bodies.

Gram(-) bacteria, S. aureus, H. pylori, E. coli strains

BL21(D3)>JM109>DH5a>M15, Archaebacteria,

nematodes and Acinetobacter sp.

B-PER Direct with Enzymes

Product # 90080

Similar to B-PER with Enzymes, but bacteria can be lysed directly in cell

culture media; ideal for screening 96-well microplate samples.

Gram(-) bacteria, S. aureus, H. pylori, E. coli strains

BL21(D3)>JM109>DH5a>M15, Archaebacteria,

nematodes and Acinetobacter sp.

Y-PER

- Yeast Protein

Extraction Reagent

Product # 78990

Easy-to-use solution gently disrupts the tough yeast cell wall in less than

20 minutes at room temperature, using a mild detergent. No mechanical

disruption needed; yields more than twice as much protein as glass

bead methods.

S. cerevisiae, Schizo-saccharomyces pombe,

C. albicans, B. subtilis, E. coli, P. pastoris, Strep.

avidinii and Acinetobacter sp.

Y-PER Plus

Product # 78998

More stringent than Y-PER, but entire formulation (including detergent)

are dialyzable.

Yeast (S. cerevisiae) and Acinetobacter sp.

M-PER

- Mammalian

Protein Extraction Reagent

Product # 78501

Highly efﬁcient total protein extraction from cultured mammalian cells;

extracts proteins in nondenatured state, enabling protein to be directly

immunoprecipitated; amine-free and fully dialyzable; adhered cells can be

directly lysed in plate or after scraping and washing in suspension.

Cultured mammalian cells, COS-7, NIH 3T3, Hepa 1-6, 293,

CHO, MDA, MB231 and FM2

P-PER - Plant Protein

Extraction Reagent

Product # 89803

Contains organic lysing reagent and two aqueous reagents, which, in

conjuction with mild mechanical agitation, effectively extract high quality

protein extracts from plant leaves, stem, root, seed and ﬂower cells without

liquid nitrogen or harsh mechanical aids, such as mortar and pestle.

Multiple plant organs (leaf, stem, root, seed and flowers);

multiple plant species (Arabidopsis, tobacco, maize,

soybeans, peas, spinach, rice and other

plant tissues); and fresh, frozen and dehydrated

plant tissues

T-PER

- Tissue Protein

Extraction Reagent

Product # 78510

Simple, easy to use reagent for extracting total protein from tissue in 1:20

(w/v) of tissue to T-PER, using centrifugation to pellet cell/tissue debris. Mild

detergent is dialyzable.

Heart, liver, kidney and brain.

I-PER - Insect Cell Protein

Extraction Reagent

Product # 89802

Optimized mild nonioinic detergent formulation provides maximum extraction

of soluble proteins from insect cells; better yield than sonication; can be used

for both suspended or adherent insect cells

Baculovirus-infected insect cells grown in

suspension or monolayer culture.

NE-PER

- Nuclear and

Cystoplasmic Extraction Kit

Product # 78833

Obtain functional concentrated nuclear extracts and cytoplasmic fractions

from mammalian cells and tissues in less than two hours, eliminating the need

for freeze/thaw cycles, Dounce homogenization, lengthy centrifugation times

and cold-room work.

Tissue: calf liver. Tissue: mouse heart, kidney, lung and

liver; Cultured cells: epithelial (HeLa), fibroid (COS-7),

kidney (NIH 3T3), liver (Hepa 1) and brain (C6).

Mem-PER

- Eukaryotic

Membrane Protein

Extraction Kit

Product # 89826

Efﬁcient, gentle reagents that solubilize and isolate membrane proteins from

mammalian and yeast cells, as well as soft and hard tissues, in less than an

hour. Minimal cross contamination (less than 10%) of hydrophilic proteins into

the hydrophobic (membrane protein) fraction

Cultured cells: brain (C6), epithelial (HeLa), fibroblasts

(NIH 3T3) and yeast (S. cerevisiae).

Subcellular Protein

Fractionation Kit

Product # 78840

Includes a combination of reagents for stepwise lysis of mammalian cells into

functional cytoplasmic, membrane, soluble nuclear, chromatin-bound, and

cytoskeletal protein fractions in a single kit; includes a stabilized nuclease

and protease inhibitors. Extracts from each compartment have less than 15%

contamination between fractions, with sufﬁcient purity for studying protein

localization and redistribution.

Cultured mammalian cells.

Mitochondrial Isolation

Kit for Cultured Cells

Product # 89874

Isolate intact mitochondria from cultured mammalian cells in approximately 40

minutes, with an optional Dounce homogenization protocol for increased yield.

Mammalian cells.

Mitochondrial Isolation

Kit for Tissues Product # 89801

Isolate intact mitochondria from soft or hard tissue in less than 60 minutes,

with an optional Dounce homogenization protocol for increased yield.

Heart, liver, kidney and brain.

Lysosome Enrichment

Kit for Tissues and Cells

Product # 89839

Uses density gradient centrifugation to separate lysozyme from

contaminating celluar structures in both mammalian cells and soft

and hard tissue.

Tissues and cultured cells.

Peroxisome Enrichment

Kit for Tissues Product # 89840

Uses density gradient centrifugation to separate peroxisome from

contaminating celluar structures in both soft and hard tissue.

Heart, liver, kidney and brain.

Nuclei Enrichment

Kit for Tissue Product # 89841

Uses density gradient centrifugation to separate nuclei from

contaminating celluar structures in both soft and hard tissue

Heart, liver, kidney and brain.

Pierce

RIPA Buffer

Product # 89900

Extracts cystoplasmic, membrane, and nuclear proteins from cultured

mammalian cells; can be used for both plated cells and cells pelleted from

suspension cultures. Protease and phosphatase inhibitors are compatible

with this formulation.

Cultured mammalian cells and cytoplasmic, membrane

and nuclear proteins.

Pierce IP Lysis Buffer

Product # 87787

Gently extracts cytoplasmic, membrane and nuclear proteins while

maintaining protein complexes for immunoprecipitation (IP), Pulldowns, and

co-IP; does not liberate DNA which can cause high viscosity.

Cultured mammalian cells.

For more details on these products, request the Cell Lysis Technical Handbook (#1601756) at www.thermoscientiﬁc.com/pierce

For more information, or to download product instructions, visit www.www.thermoscientiﬁc.com/pierce

Protein Puriﬁcation Accessories

Protease and Phosphatase Inhibitors

for Protein Puriﬁcation

Thermo Scientiﬁc Halt Protease and Phosphase Inhibitors

Our broad-spectrum protease and phosphatase inhibitor cocktails

and individual protease inhibitors accommodate speciﬁc and

general needs in cell lysis and protein extraction methods.

Highlights

• Halt Protease Inhibitor Cocktails target serine, cysteine and

aspartic acid proteases and aminopeptidases. Metalloproteases

are inhibited by the optional addition of EDTA. Individual protease

inhibitors targeting separate classes of

proteases are also available for custom cocktail development.

• Halt

™

Phosphatase Inhibitor Cocktails contain chemical com-

pounds that target serine/threonine and tyrosine phosphatases.

• The Halt Protease and Phosphatase Inhibitor Cocktail prevents

protein degradation and preserves phosphorylation simultaneously,

providing protection that is similar to the individual cocktails.

For a complete listing of the Halt Protease and Phosphatase

Inhibitors at www.thermoscientiﬁc.com.

Buffers for Protein Puriﬁcation

Thermo Scientiﬁc BupH Pack Dry Blend Buffers

BupH

Packs are pre-blended and pre-measured dry mixtures of

commonly needed buffers that are easy to prepare; simply empty

contents of foil envelope pack into a beaker, add ultrapure water,

and stir to dissolve. The packs eliminate weighing time and tedious

pH adjustments. BupH Pack Dry Blend Buffers are offered for use in

common laboratory techniques and to support other Pierce Protein

Research Products.

Highlights:

• Convenient – dissolve contents of one envelope in 500ml of water

and the buffer is ready to use

• Save time and trouble – no weighing, no pH adjustment, no need

to stock individual components and no need to make and store

large volumes of stock solution in advance of daily needs

• Long shelf life – stocking and storage as dry packs eliminates

concerns about long-term stability of stock solutions

• Eliminate variables – our quality control ensures that every pack

will yield the same, consistent buffer

Ordering Information

Product #

Description

Pkg. Size

Applications

Formulation of each pack

after reconstitution

U.S.

Price

28384 Borate Buffer

40 packs

Protein modiﬁcation procedures that

require amine-free buffer at alkaline pH

500mL of 50mM borate, pH 8.5

$132

28382 Carbonate-Bicarbonate Buffer

40 packs

Microplate protein and antibody coating

for ELISA or RIA

500mL of 0.2M carbonate-bicarbonate, pH 9.4

$125

28388 Citrate-Carbonate Buffer

10 packs

Protein immobilization to UltraLink

Biosupport (Product # 53110)

100mL of 0.6M sodium citrate,

0.1M sodium carbonate, pH 9

$ 64

28386 Citrate-MOPS Buffer

10 packs

Protein immobilization to UltraLink

Biosupport (Product # 53110)

100mL of 0.6M sodium citrate,

0.1M MOPS, pH 7.5

$ 58

28390 MES Buffered Saline

10 packs

Crosslinking using carbodiimide

(EDC, Product # 22980)

500mL of 0.1M MES, 0.9% NaCl, pH 4.7

$112

28372 Phosphate Buffered Saline (PBS)

40 packs

Crosslinking and biotinylation requiring

amine-free buffer

500mL of 0.1M sodium phosphate,

0.15M NaCl, pH 7.2

$135

28374 Modiﬁed Dulbecco’s PBS

40 packs

Wash buffers and antibody diluents

for ELISA, Western blotting and other

immunoassays

500mL of 8mM sodium phosphate,

2mM potassium phosphate, 0.14M NaCl,

10mM potassium chloride, pH 7.4

$ 96

28379

28376

Tris Buffered Saline

10 packs

40 packs

Wash buffers and antibody diluents

for ELISA, Western blotting and other

immunoassays

500mL of 25mM Tris,

0.15M NaCl, pH 7.2

$ 61

$134

28378 Tris-Glycine-SDS Buffer

40 packs

Running buffer for Tris-Glycine gel

electrophoresis

500mL of 25mM Tris, 192mM glycine,

0.1% SDS, pH 8.3

$120

28398 Tris-HEPES-SDS Running Buffer 10

10 packs

Running buffer for Tris-HEPES electrophoresis

with Precise

™

Precast Gels

500mL of 100mM Tris, 100mM HEPES,

3mM SDS, pH 8±0.25

$ 36

28380 Tris-Glycine Transfer Buffer

40 packs

Running buffer for gel to membrane

electrophoretic transfer

500mL of 25mM Tris, 192mM glycine,

pH 8 (use 20% methanol to dissolve)

$120

Need a larger quantity?

Call our Bulk and Custom

department at 1-800-874-3723

or +1 815-968-0747 ext. 300 for

pricing and information. Or, visit

www.thermoscientiﬁc.com/protein-custom

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch ofﬁce or distributor.

Thermo Scientiﬁc Concentrated Liquid Buffers

Save counter space and time.

Pierce 10X and 20X Concentrated Buffers are ready to use without

having to reconstitute with ultrapure water and, in the case of

Tris-Glycine Buffer, methanol. Buffers are designed for use in

dialysis, cross-linking, enzyme assays, ELISAs, immunohistochem-

istry, protein plate-coating, biotinylation and other applications.

Keep our concentrated buffers close at hand to cover your lab’s

research needs.

Highlights:

• Easy to use – no packets to open and no powder to dissolve

• Increased accuracy – eliminates the possibility of powder

remaining in a packet

• Saves time – 10X or 20X concentration eliminates time spent

waiting for powder to dissolve

• Saves space – storage as concentrated stock minimizes bench

space needed for large volume solutions

PBS

Tris-Glycine-SDS

Tris-Hepes-SDS

Ordering Information

Product #

Description

Pkg. Size

Applications

1X Formulation

U.S.

Price

28341 20X Borate Buffer

500mL

Ideal for protein modiﬁcation procedures

requiring amine free buffer at alkaline PH

500mL of 50mM borate, pH 8.5

$68

28344 20X Modiﬁed Dulbecco's

PBS Buffer

500mL

Used for wash buffers and antibody diluents

in applications such as ELISA, Western

blotting and other immunoassays

8mM Sodium Phosphate,

2mM Potassium Phosphate,

0.14M NaCl, 100mM KCl, pH 7.4

$68

28346 20X Modiﬁed Dulbecco's

PBS Tween-20 Buffer

500mL

A wash buffer for ELISA, Western and

other Immunoassays as well as a blocking

buffer for plate based assays

8mM Sodium Phosphate,

2mM Potassium Phosphate,

0.14M NaCl, 100mM KCl,

0.05% Tween-20, pH 7.4

$68

28348 20X Phosphate Buffered Saline

500mL

Its ionic strength makes it ideal for

crosslinking and biotinylation requiring

amine free buffer

0.01M Sodium Phosphate,

0.15M NaCl, pH 7.5

$68

28352 20X PBS Tween-20 Buffer

500mL

A wash buffer for ELISA, Western and other

Immunoassays as well as a blocking buffer

for plate based assays

0.01M Sodium Phosphate,

0.15M NaCl, 0.05% Tween-20, pH 7.5

$68

28354 20X TAE Buffer

500mL

Historically the most common buffer used

for agarose gel electrophoresis in the

analyses of nucleic acids

0.04M Tris, 0.04M Acetate,

0.001M EDTA, pH 8.2-8.4

$68

28355 10X TBE Buffer

Often used for agarose gel electrophoresis

in the analysis of nucleic acids

0.089M Tris, 0.089 M Borate,

0.002M EDTA, pH 8.2-8.4

$65

28358 20X TBS Buffer

500mL

Used for wash buffers and antibody diluents

in applications such as ELISA, Western

blotting and other immunoassays

25mM Tris, 0.15M NaCl, pH 7.2

$68

28360 20X TBS Tween-20 Buffer

500mL

A wash buffer for ELISA, Western and other

Immunoassays as well as a blocking buffer

for plate based assays

25mM Tris, 0.15M NaCl,

0.05% Tween-20, pH 7.5

$68

28362 20X Tris/HEPES/SDS Buffer

A running buffer for Tris-HEPES

electrophoresis with Precise Precast Gels

0.025 M Tris, 0.192 M Glycine,

0.1% SDS, pH 8.5

$45

28363 10X Tris-Glycine Buffer

Great for electrophoretic transfer from

gel to membrane

0.025M Tris, 0.192M Glycine, pH 8.5

$45

28368 20X Tris/HEPES/SDS Buffer

500mL

A running buffer for Tris-HEPES

electrophoresis with Precise Precast Gels

0.1M Tris, 0.1M HEPES,

3mM SDS, pH 8+0.25

$68

For more information, or to download product instructions, visit www.www.thermoscientiﬁc.com/pierce

Spin Cups and Columns

Thermo Scientiﬁc Pierce Spin Columns are convenient tools for

manipulating small volumes of afﬁnity supports (5–500μL) for

protein puriﬁcation. Add the afﬁnity resin and sample to one of

the columns, use a microcentrifuge to efﬁciently wash away

contaminants and elute your puriﬁed sample without losing any

resin in the process. Spin columns allow you to afﬁnity-purify

more protein in less time!

Highlights:

• Efﬁcient washing of samples means fewer washes are needed

to remove contaminating proteins

• Efﬁcient elution of samples means more antigen and

co-precipitated proteins are recovered

• No resin loss means more consistent IP and co-IP results

• No need to decant supernatant from IP or co-IP pellet

• Spin protocols drastically reduce the time required for IPs and co-IPs

• Low protein-binding polypropylene column construction

minimizes nonspeciﬁc binding

Spin Cups – Paper Filter

Paper ﬁlter with collection tubes.

Highlights:

• Paper ﬁlters are resistant to clogging

from cellular debris

• Column Volume: 600μL

• Resin Volume: 20–300μL

• Filter Type: Paper, ~10μm pore size

• Cap Type: Collection tube cap ﬁts onto inserted spin cup

Spin Cups – Cellulose Acetate Filter

Cellulose acetate ﬁlter with collection tubes.

Highlights:

• Used in our IP and Co-IP Kits

• Column Volume: 800μL

• Resin Volume: 20–400μL

• Filter Type: Cellulose acetate, 0.45μm pore size

• Cap Type: Collection tube cap ﬁts onto inserted spin cup

Spin Columns – Screw Cap

Screw cap with Luer-Lok

Adaptors.

Highlights:

• Luer-Lok Adaptors allow these columns

to be used for syringe-based puriﬁcations

• Column Volume: 900μL

• Resin Volume: 20–400μL

• Filter Type: Polyethylene, ~10μm pore size

• Small & large frit options for different sample sizes

• Cap Type: O-ring screw top caps; press-in bottom plugs

Spin Columns – Snap Cap

Snap cap with collection tubes.

Highlights:

• Used in the Cell Surface Protein Isolation

and Glycoprotein Isolation Kits

• Column Volume: 1,000μL

• Resin Volume: 20–500μL

• Filter Type: Polyethylene, ~30μm pore size

• Cap Type: Snap cap on column (no cap on collection tube);

press-on bottom caps

Micro-Spin Columns

Highlights:

• Column Volume: 400μL

• Resin Volume: 5–100μL

• Filter Type: Polyethylene, ~30μm pore size

• Cap Type: O-ring screw top caps; press-on

bottom caps

Ordering Information

Product #

Description

Pkg. Size

U.S.

Price

69700 Spin Cups – Paper Filter

Includes cups and collection tubes

50/pkg

$109

69715 Microcentrifuge Tubes

Collection Tubes for Product # 69700

72/pkg

$ 32

69702 Spin Cups – Cellulose Acetate Filter

Includes cups and collection tubes

50/pkg

$108

69720 Microcentrifuge Tubes

Collection Tubes for Product # 69702

72/pkg

$ 32

69705 Spin Columns – Screw Cap

with Luer-Lok Adaptors

Includes: Spin Columns, Screw Caps and Column Plugs

Luer-Lok Adaptors

Large Frits (6.8mm diameter 10μm pore size)

Small Frits (2.7mm diameter 10μm pore size)

Large and Small Frit Tools

Kit

25 each

5 each

25 each

1 each

$ 99

69725 Spin Columns – Snap Cap

with Collection Tubes

Includes: Spin Columns and Bottom Caps

Collection Tubes

Kit

50 each

100 each

$109

89879 Micro-Spin Columns

50/pkg

$ 55

Protein Puriﬁcation Accessories

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch ofﬁce or distributor.

Disposable Plastic Columns

Automatic “stop-ﬂow” action provided by porous polyethylene

discs prevents column beds from drying out.

Highlights:

• Supplied complete with porous polyethylene discs, stoppers, end caps

• Compatible with most types of aqueous buffer eluents commonly

used in chromatography

• Can be pre-packed and stored until needed

Ordering Information

Product #

Description

Pkg. Size

U.S.

Price

29920 Disposable Polystyrene Columns

Ideal for packing 0.5–2.0mL bed volumes.

100/pkg

$144

29922 Disposable Polypropylene Columns

Ideal for packing 1–5mL bed volumes.

100/pkg

$174

29924 Disposable Polypropylene Columns

Ideal for packing 2–10mL bed volumes.

100/pkg

$190

29923 Disposable Polypropylene Funnels

Buffer reservoirs that ﬁt Product #’s 29920,

29922 and 29924.

50/pkg

$109

29925 Disposable Column Trial Pack

Includes accessories plus two each of Product #’s

29920, 29922 and 29924 and one of Product # 29923.

Trial Pack

$ 63

Centrifuge Columns

Efﬁciently handle a wide variety of resin volumes for afﬁnity

puriﬁcation! Thermo Scientiﬁc Pierce Centrifuge Columns are

convenient tools for handling 40μL–10mL of an afﬁnity puriﬁcation

support. Add the afﬁnity resin to one of the polypropylene columns,

remove the twist-off bottom and allow the resin to pack itself.

Then add your sample and allow it to bind to the support. Use a

centrifuge to efﬁciently wash away any contaminants and elute

your puriﬁed protein.

Pierce Centrifuge Columns allow you to use a spin-column format

in addition to traditional gravity ﬂow to reduce the time required for

column washing and elution. This accelerates sample processing

time and makes multiple-sample processing possible. Centrifuge

columns allow you to afﬁnity-purify more protein in less time!

Centrifuge columns are made from low protein-binding

polypropylene for compatibility with protein puriﬁcation, and

they ﬁt into standard centrifuge tubes for use in any centrifuge.

Applications for Centrifuge Columns:

• Afﬁnity puriﬁcation/afﬁnity chromatography

• Immunodepletion

• Spin desalting

0.8mL Centrifuge Columns

Highlights:

• Total Volume: 800μL

• Resin Volume: 40–400μL

• Filter Type: Polyethylene, ~30μm pore size

• Receiver Tube: Fits standard microcentrifuge tubes

(e.g., Product # 69720)

• Cap Type: O-ring screw-top cap

• Twist-off bottom

2mL Centrifuge Columns

Highlights:

• Total Volume: 5mL (2mL resin bed, 3mL reservoir)

• Resin Volume: 2mL

• Filter Type: Polyethylene, ~30μm pore size

• Receiver Tube: Fits standard 15mL conical

centrifuge tubes

• Cap Type: Screw-top cap

• Twist-off bottom and press-on cap to reseal

5mL Centrifuge Columns

Highlights:

• Total Volume: 8mL (5mL resin bed, 3mL reservoir)

• Resin Volume: 5mL

• Filter Type: Polyethylene, ~30μm pore size

• Receiver Tube: Fits standard 15mL conical

centrifuge tubes

• Cap Type: Screw-top cap

• Twist-off bottom and press-on cap to reseal

10mL Centrifuge Columns

Highlights:

• Total Volume: 22mL (10mL resin bed, 12mL reservoir)

• Resin Volume: 10mL

• Filter Type: Polyethylene, ~30μm pore size

• Receiver Tube: Fits standard 50mL conical

centrifuge tubes

• Cap Type: Screw-top cap

• Twist-off bottom and press-on cap to reseal

Ordering Information

Product #

Description

Pkg. Size

U.S.

Price

89868 Centrifuge Columns, 0.8mL capacity

Includes: Pierce Centrifuge Columns

Screw Caps

Kit

50 each

$ 62

89869 Centrifuge Columns, 0.8mL capacity

Includes: Pierce Centrifuge Columns

Screw Caps

Kit

4 x 50 each

$192

89896 Centrifuge Columns, 2mL capacity

Includes: Pierce Centrifuge Columns

Screw Caps and Tips

Kit

25 each

$ 39

89897 Centrifuge Columns, 5mL capacity

Includes: Pierce Centrifuge Columns

Screw Caps and Tips

Kit

25 each

$ 44

89898 Centrifuge Columns, 10mL capacity

Includes: Pierce Centrifuge Columns

Screw Caps and Tips

Kit

25 each

$ 49

69707 Column Extender

Fits 89896, 89897 and 89898. Increases column

capacity by 35mL

10 each

$ 28

5ml

4ml

3ml

2ml

1ml

10ml

9ml

8ml

7ml

6ml

5ml

4ml

3ml

2ml

1ml

For more information, or to download product instructions, visit www.thermoscientiﬁc.com/pierce

Fusion Protein Puriﬁcation

Cultures of E. coli or Picchia are common vehicles for protein

expression. They are low cost and low maintenance platforms

which can be easily scaled up to deliver protein at milligram to

gram yields. These microoganisms are also very easy to trans-

form with a DNA vector containing the gene of interest. Typically,

researchers use common expression vectors which possess the

proper promoter elements for expression and inclusion of an

afﬁnity tag sequence. The afﬁnity tag sequence is cloned in frame

with the DNA sequence of the target protein, and will ﬂank either

the N- or C-terminus. The two most common afﬁnity tags are

polyhistidine (6xHis) and glutathione-S-trasferase (GST).

Polyhistidine Puriﬁcation

Cobalt-histidine binding.

The polyhistidine tag is a sequence of ﬁve to nine histidine amino

acids attached to the terminus of a target protein. The polyhistidine

tag is puriﬁed using immobilized metal afﬁnity chromatography

(IMAC). For histidine tag puriﬁcation, either nickel or cobalt is

immobilized onto a solid chromatography resin. While the two

metals can be used interchangeably, typically nickel has a higher

binding capacity whereas cobalt binds less non-speciﬁc protein to

deliver a purer ﬁnal protein.

IMAC resins work by charge interactions with the nitrogen atoms

on the histidine amino acid side chain to bind and immobilize the

histidine-tagged protein from a cell lysate. The incorporation of

multiple histidine residues as an afﬁnity tag is designed to improve

this charge association. Because IMAC afﬁnity for histidine resi-

dues is not dependent on the secondary structure of the protein,

IMAC puriﬁcation can be performed under denaturing conditions.

IMAC puriﬁcation is, however, sensitive to pH and the presence of

chelators and reducing agents. See Table 1 for a list of common

interfering agents and their concentration tolerance for both nickel

and cobalt resin.

Fusion Protein Puriﬁcation

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch ofﬁce or distributor.

Nickel-histidine binding.

Table 1. Common interfering agents for Ni-NTA and cobalt resins.

Reagent Tolerance for Ni-NTA resin Tolerance for Cobalt resin

ß-Mercaptoethanol 10mM 10mM

CHAPS 1% 1%

Ethanol 30% 30%

Ethylene glycol 30% 30%

HEPES 50mM 50mM

Glycerol 20% 20%

Guanidinium 6M 6M

Imididazole 500mM 200mM at pH 7.0-8.0 for elution

KCl 500mM 500mM

MES 20mM 20mM

MOPS 50mM 50mM

NaCl 1.0M 1.0M

NP-40 1% 1%

SDS 1% 1%

TRIS 50mM 50mM

Triton

-X-100 <1% <1%

Urea 8M 8M

Once immobilized, imidazole is used to disrupt the charge

attractions between the immobilized metal afﬁnity chromatography

resin and the histidine-tagged protein. The eluted histidine-tagged

protein can be easily cleaned-up using a desalting column or

dialysis cassette to remove imidazole. Often trace amounts of

imidazole are included in the protein binding and wash steps

to compete away binding of endogenous proteins with multiple

histidines.

Glutathione-S-transferase Puriﬁcation

GST binding.

GST is a 26kDa endogenous enzyme found in both prokaryotes

and eukaryotes. In the cell GST detoxiﬁes reactive oxygen species

such as free radicals and peroxides. GST performs this task by

neutralizing reactive oxygen species with the antioxidant glutathione

(GSH). GSH is an endogenous tripeptide (Glu-Cys-Gly) containing

a cysteine residue, whose sulfhydryl side chain functions as a

reducing agent.

GST makes an ideal afﬁnity tag because of its strong binding to

reduced glutathione. GST possess numerous tyrosine residues in

the in the GSH binding pocket, and one of these tyrosines hydrogen

bonds to the substrate glutathione forming a stable complex.

In vivo, GST would transfer glutathione to a reactive oxygen

species and neutralize it.

As a research tool, scientists have taken advantage of the strong

interaction between GST and GSH to selectively extract recom-

binant proteins. In this strategy, glutathione is immobilized onto

a solid support (typically an agarose bead) and the amino acid

sequence for the enzyme GST is cloned into either the C- or

N-terminus of the target gene (commonly using the pGEX

-series

of expression vectors). After binding of the GST-tagged fusion

protein to the immobilized glutathione agarose, excess reduced

glutathione is introduced (typically between 10-50mM) to compete

off the target protein. Again, simple dialysis or desalting columns

can be used to clean up the ﬁnal GST-tagged protein. For reasons

that have not been fully characterized in the literature, the struc-

ture of the GST fusion tag often degrades upon denaturation and

reduction for protein gel electrophoresis (e.g., SDS-PAGE). As a

result, electrophoresed samples often appear as a ladder of lower

MW bands below the full-sized fusion protein.

In contrast to polyhistidine puriﬁcation, GST puriﬁcation requires

the target protein maintain native tertiary structure. Additionally,

the GST tag is quite large (26kDa) compared to the six histidines

which comprise a typical polyhistidine tag. To circumvent the

problems associated with a 26kDa appendage, selective proteases

are used to cleave the GST tag from the GST-fusion protein. These

proteases include Factor Xa (Product # 32520) or thrombin.

For more information, or to download product instructions, visit www.thermoscientiﬁc.com/pierce

His-Tagged Protein Puriﬁcation Resin

A cost-effective Ni-NTA resin is now available.

The expression and puriﬁcation of

recombinant proteins is central to protein

regulation, structure and function stud-

ies. The majority of recombinant proteins

are expressed as fusions with short

afﬁnity tags with the most popular being

the polyhistidine (6xHis) tag. The method

used to purify recombinant His-tagged

proteins is immobilized metal afﬁnity

chromatography (IMAC), consisting of

chelating resins charged with either

nickel or cobalt ions that coordinate

with the histidine side chains. The new

Thermo Scientiﬁc HisPur Ni-NTA Resin

complements the HisPur

™

Cobalt Resin. Ni-NTA resins are the most

common IMAC resin choice for 6xHis-tagged protein puriﬁcations

because of the four metal-binding sites on the chelate, which

enables high-protein binding and low-metal ion leaching.

Highlights:

• High capacity – bind up to 60mg of 6xHis-tagged protein per

milliliter of resin

• Versatile – purify proteins using native or denaturing conditions

• Compatible – use with Thermo Scientiﬁc Cell Lysis Reagents

and a variety of buffer additives

• Flexible – available in multiple formats, including bulk resin, spin

columns and chromatography cartridges

• Cost-effective – reuse the same batch of resin at least ﬁve times

• Easy to use – pre-formulated buffers available for kit formats

His-tagged green ﬂuorescent protein (GFP) was recovered in

similar or greater purity and yield compared to other commercially

available nickel-chelated resins (Figure 1). HisPur Ni-NTA Resin

is also cost-effective because it can be used at least ﬁve times

without loss in performance (Figure 2). The resin is amenable to

chromatography-cartridge format, resulting in a sharp elution

peak evident in the chromatogram (Figure 3). When HisPur Ni-NTA

Resin is compared to HisPur Cobalt Resin (Product # 90090) and

Ni-IDA resin using the batch-bind method, the results depend on

protein expression level (Table 2 and Figure 4). When purifying a

low-expression protein, such as 6xHis-AIF2, there is a dramatic

difference in purity. HisPur Cobalt Resin yields the purest protein

followed by HisPur Ni-NTA Resin with Ni-IDA being the least pure.

When purifying 6xHis-GFP, a high expresser, there was minimal

difference between HisPur Ni-NTA and Cobalt Resins. Similar

results were obtained using spin puriﬁcation (data not shown).

100

150

250

250kDa

HisPur Ni-NTA

Supplier Q

Supplier C

Ni-IDA

6xHis-GFP

Figure 1. Thermo Scientiﬁc HisPur Ni-NTA resin is comparable to or

performs better than other suppliers’ nickel resins. Bacterial lysate

(12mg total protein) containing over-expressed 6xHis-green ﬂuorescent

protein (GFP) was applied to HisPur Ni-NTA Resin (200μL) and puriﬁed by

the batch-bind method as described. The same amount of total protein

was applied to Supplier Q, Supplier C and Ni-IDA resins per the manu-

facturers’ instructions. Gel lanes were normalized to equivalent volume.

M = molecular-weight marker and L = lysate load.

Fusion Protein Puriﬁcation

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch ofﬁce or distributor.

First Use

L FT W E

Fifth Use

L FT W E

100

150

kDa

6xHis-

Protein L

Figure 2. Thermo Scientiﬁc HisPur Ni-NTA Resin can be used at least ﬁve

times without losing performance. Bacterial lysate (20mg total protein) con-

taining over-expressed 6xHis-Protein L was applied to HisPur Ni-NTA Resin

(200μL) and puriﬁed by the batch-bind method as described. Before each

reuse, the resin was washed with 20mm MES buffer, 0.1 M NaCl; pH 5 (1mL)

and followed by a water wash (1mL). Gels lanes were normalized to equivalent

volume. M = molecular-weight marker, L = lysate load, FT = ﬂow-through and

E = elution.

280 nm

485 nm

500

1000

1500

2000

2500

3000

3500

mAU

Flow-through

Wash

0 20 40 60 80 100 120mL

6xHis-GFP

Figure 3. Thermo Scientiﬁc HisPur Ni-NTA Chromatography Cartridges

provide an automatable solution for obtaining high-purity proteins. Bacterial

lysate (140mg total protein) containing over-expressed 6xHis-GFP was diluted

with equilibration buffer and applied to a HisPur Ni-NTA Chromatography

Cartridge at a ﬂow rate of 1mL/min. The cartridge was washed with PBS,

60mm imidazole until the baseline absorbance was reached. 6xHis-GFP was

eluted with PBS, 300mm imidazole. Elution was monitored at 280nm (blue line)

and 485nm (red line).

Table 2. Elution fractions were analyzed for protein content using the Thermo

Scientiﬁc Coomassie Plus (Bradford) Protein Assay Kit (Product # 23236).

Purity was determined by analyzing the stained SDS-PAGE gel (Figure 4)

with densitometry software.

Sample Resin Total yield (mg) Purity

6xHis-AIF2

HisPur Ni-NTA 0.5 32%

Ni-IDA 0.5 25%

HisPur Cobalt 0.4 49%

6xHis-GFP

HisPur Ni-NTA 0.8 90%

Ni-IDA 0.6 52%

HisPur Cobalt 0.7 91%

100

150

250

kDa

HisPur Ni-NTA

Ni-IDA

HisPur Cobalt

HisPur Ni-NTA

Ni-IDA

HisPur Cobalt

6xHis-GFP

6xHis-AIF2

Figure 4. Thermo Scientiﬁc HisPur Cobalt Resin produces the most pure

results. Bacterial lysate containing over-expressed 6xHis-AIF2 (6mg total

protein) or 6xHis-GFP (4mg total protein) was applied to HisPur Ni-NTA Resin

(200μL) and puriﬁed by the batch-bind method as described. The same amount

of total protein was applied to Ni-IDA and HisPur Cobalt Resins and puriﬁed

as described in the instructions. Gels lanes were normalized to equivalent

volume. M = molecular-weight marker, L = lysate load.

The HisPur Ni-NTA Resin offers a cost-effective alternative to

other commercially available nickel-IMAC resins, without compro-

mising yield, purity or performance. The HisPur Ni-NTA and Cobalt

Resins are available in multiple formats to accommodate a variety

of applications and puriﬁcation volumes.

Ordering Information

Product # Description Pkg. Size

U.S.

Price

88221 HisPur Ni-NTA Resin

10mL

$ 85

88222 HisPur Ni-NTA Resin

100mL

$ 579

88223 HisPur Ni-NTA Resin

500mL

$2275

88224 HisPur Ni-NTA Spin Columns, 0.2mL

25 columns

$ 155

88225 HisPur Ni-NTA Spin Columns, 1mL

5 columns

$ 99

88226 HisPur Ni-NTA Spin Columns, 3mL

5 columns

$ 150

88227 HisPur Ni-NTA Puriﬁcation Kit

, 0.2mL

25 columns

$ 269

88228 HisPur Ni-NTA Puriﬁcation Kit

, 1mL

5 columns

$ 253

88229 HisPur Ni-NTA Puriﬁcation Kit

, 3mL

5 columns

$ 269

90098 HisPur Ni-NTA Chromatography

Cartridges, 1mL

5 cartridges

$ 155

90099 HisPur Ni-NTA Chromatography

Cartridges, 5mL

2 cartridges

$ 200

§ For complete ordering information and kit components, please visit

www.thermoscientiﬁc.com/pierce

For more information, or to download product instructions, visit www.thermoscientiﬁc.com/pierce

Cobalt Resin, Spin Columns

and Chromatography Cartridges

Speciﬁc, fast and gentle puriﬁcation of His-tagged proteins.

The preferred method for purifying recombinant His-tagged

proteins is immobilized metal afﬁnity chromatography (IMAC).

Traditionally, chelating chromatography resins are charged with

either nickel or cobalt ions that coordinate with the histidine side

chains in the 6xHis-tag. HisPur Cobalt Resin is a tetradentate

chelating resin charged with cobalt that binds His-tagged proteins

with high speciﬁcity and releases them under lower imidazole

concentrations than required with nickel resins (see Figure 5).

HisPur Cobalt Resin can be used to obtain high-purity proteins with

no metal contamination.

The HisPur Chromatography Cartridges are convenient, reliable

and ready-to-use pre-packed devices for the isolation of proteins

in solution and puriﬁcation of His-tagged fusion proteins. The

HisPur Cartridges are compatible with automated LC instrumen-

tation, such as the ÄKTA

and BioLogic Systems, and adapt to

manual syringe processing.

Wash Elution

16.5

110

210

kDa

M F 1 2 3 1 2 3 4 5 L

6xHis-GFP

Thermo Scientiﬁc HisPur Cobalt Resin is speciﬁc for His-tagged proteins and

allows mild, efﬁcient elutions. Bacterial lysate (1.0mg total

protein) was applied to a 200μL bed volume of HisPur Cobalt Resin in a spin

column. Gel lanes were normalized to equivalent volume. M = Molecular

Weight Markers (Product # 26691), L = lysate load and F = ﬂow-through.

Highlights:

• High purity – obtain pure protein without optimizing imidazole

washing conditions

• Speciﬁcity – cobalt:chelate binding core binds fewer contaminants,

resulting in lower background than nickel (see Table 3)

• Low metal leaching – no metal contamination in eluted sample

• Versatility – purify proteins under native or denaturing conditions;

compatible with cell lysis reagents and a variety of buffer additives

• Flexibility – available as bulk resin or predispensed columns

compatible with both spin and gravity-ﬂow formats

• Cost effective – reuse or discard

• Superior – performs better than other commercially available

IMAC Resins (see Figure 5)

GFP β-gal Protein L

2 3 4 5 6 L 1 2 3 4 5 6 L 1 2 3 4 5 6 L

Figure 5. Thermo Scientiﬁc HisPur Cobalt Resin outperforms other IMAC

resins. Comparable yield and higher purity obtained with HisPur Cobalt Resin

(lane 1) compared to other IMAC resins (lanes 2 to 6). Cell lysates containing

over-expressed recombinant 6xHis-tagged protein were prepared in B-PER

Bacterial Protein Extraction Reagent (Product # 78243) and Protease Inhibitors

(Product # 78410). Protein concentrations were determined by Coomassie

Plus Protein Assay (Product # 23238). E. coli lysates containing over-expressed

His-tagged GFP, β-galactosidase or Protein L were applied to 0.2mL bed

volumes of each IMAC resin in spin column format. Binding, wash and elution

buffers were prepared and used per each manufacturers’ instructions. The ﬁrst

elution fraction for each IMAC resin was analyzed by SDS-PAGE and protein

purity determined by densitometry. Gel lanes were normalized to equivalent

volume. Lanes: 1= HisPur Cobalt Resin, 2= supplier C cobalt resin, 3= supplier

S cobalt resin, 4= supplier G nickel resin, 5= supplier Q nickel resin, 6= Ni-IDA

and L= lysate load.

Fusion Protein Puriﬁcation

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch ofﬁce or distributor.

Table 3. Thermo Scientiﬁc HisPur Cobalt Resin yields more His-tagged protein and higher purity than other Co

and Ni

IMAC Resins.

His-GFP

His-β-Gal

His-Protein L

Yield (μg)* Purity (%)** Yield (μg)* Purity (%)** Yield (μg)* Purity (%)**

Thermo Scientiﬁc HisPur Cobalt Resin 298 87 78 93 42 77

Supplier C Cobalt Resin 206 78 26 90 35 76

Supplier S Cobalt Resin 211 85 27 65 38 77

Supplier G Nickel Resin 239 84 42 83 29 68

Supplier Q Nickel Resin 242 85 24 48 30 72

-IDA Resin 70 37 6 16 17 46

* Recovered from a 5mg total protein load (total protein yield x purity). ** Purity of the ﬁrst elution fraction.

HisPur Cobalt Cartridge Performance Data:

1000

2000

3000

4000

5000

6000

0 5 10 15 20 25

Chromatography Progress (mL)

Flow-through Wash Elution

Absorbance 280nm (mAU)

Flow-through (FT)M S Wash Elution

Electrophoresed Fractions

Chromatography Profile

Puriﬁcation of 6xHis-GFP from E. coli lysate using a Thermo Scientiﬁc HisPur

Cobalt Cartridge. His-tagged green ﬂuorescent protein (GFP) was extracted

from E. coli using Thermo Scientiﬁc B-PER Bacterial Protein Extraction

Reagent in Phosphate Buffer (Product # 78266) containing Halt Protease

Inhibitor Cocktail, EDTA-Free (Product # 78415). The lysate was diluted 1:1

with equilibration/wash buffer (50mM sodium phosphate, 300mM sodium

chloride, 10mM imidazole, pH 7.4) and applied to a Thermo Scientiﬁc HisPur

Cobalt Chromatography Cartridge at a ﬂow rate of 0.3mL/min. The cartridge

was washed with equilibration/wash buffer until the baseline absorbance

at A

280

was reached. His-tagged GFP was eluted (50mM sodium phosphate,

300mM sodium chloride, 150mM imidazole; pH 7.4) and selected fractions

were analyzed by SDS-PAGE and Thermo Scientiﬁc GelCode Blue Stain

Reagent (Product # 24592). M = MW Marker; S = non-fractionated lysate;

FT = ﬂow-through.

Ordering Information

Product #

Description

Pkg. Size

U.S.

Price

89967 HisPur Cobalt Spin Columns, 0.2mL

25 columns

$ 160

89968 HisPur Cobalt Spin Columns, 1mL

5 columns

$ 105

89969 HisPur Cobalt Spin Columns, 3mL

5 columns

$ 155

89964 HisPur Cobalt Resin

10mL bottle

$ 88

89965 HisPur Cobalt Resin

100mL bottle

$ 600

89966 HisPur Cobalt Resin

500mL bottle

$2347

90090 HisPur Puriﬁcation Kit, 0.2mL

25 columns

$ 277

90091 HisPur Puriﬁcation Kit, 1mL

5 columns

$ 262

90092 HisPur Puriﬁcation Kit, 3mL

5 columns

$ 277

90093 HisPur Chromatography Cartridges

5 x 1mL

$ 160

90094 HisPur Chromatography Cartridges

2 x 5mL

$ 267

For more information, or to download product instructions, visit www.thermoscientiﬁc.com/pierce

Fusion Protein Puriﬁcation

High-quality puriﬁcation of GST-fusion proteins

Multiple formats available to meet your puriﬁcation needs

Puriﬁcation of glutathione S-transferase (GST) fusion proteins

using glutathione agarose beads provides one-step, high-purity

afﬁnity puriﬁcation. The Thermo Scientiﬁc Pierce Glutathione

Agarose is composed of 6% crosslinked beaded agarose with

glutathione (GSH) immobilized by its central sulfhydryl. GST-fusion

proteins are puriﬁed with high yield because of the 12-atom GSH

linker that

minimizes steric hindrance. The Pierce Glutathione Agarose is

available in resin slurry packages, spin columns, complete

puriﬁcation kits and FPLC-ready chromatography cartridges.

Highlights:

• High capacity – binds at least 40mg of pure GST per milliliter

of resin

• Cost-effective – resin is economically priced and can be reused

at least ﬁve times without reduction in performance

• High yield and purity – each milliliter of resin can purify ≥ 10mg

of GST-tagged protein from lysates with > 90% purity

• Compatible – validated and effective with Thermo Scientiﬁc

Cell Lysis Reagents

• Versatile – purify lysates or pulldown protein-protein interactions

• Flexible – available in multiple formats

Glutathione S-transferase and polyhistidine are the most

commonly used protein tags for afﬁnity puriﬁcation. GST

produces cleaner puriﬁcations than histidine tags and stabilizes

overexpressed fusion proteins. GST binds speciﬁcally to reduced

GSH in near-neutral, nondenaturing conditions. The bound

GST-tagged protein is easily dissociated (eluted) from GSH resin

by competitive displacement with a buffer containing free, reduced

GSH. Alternatively, if the fusion protein is designed with a protease

cleavage site, it may be released (cleaved) from the column at the

GST tag junction using thrombin, HRV 3C protease or Factor Xa.

The Pierce Glutathione Agarose is highly effective for purifying a

variety of GST-fusion proteins (Figure 6). The purity and yield of

GST-fusion protein recovery is comparable to or better than

glutathione resins from other suppliers (Figure 7). The resin is

composed of 6% crosslinked beaded agarose, making it a

structurally stable and resilient afﬁnity support that enables

repeated uses. The Pierce Glutathione Agarose is also amenable

to chromatography cartridge format, producing baseline

resolution and a sharp elution peak (Figure 8) with no visible

resin bed compression.

GST-Pak1

kDa M L FT W1 W2 W3 E1 E2 E3

210

110

16.5

GST-ERK

M L FT W1 W2 W3 E1 E2 E3kDa

210

110

16.5

GST-Bid

M L FT W1 W2 W3 E1 E2 E3kDa

210

110

16.5

Figure 6. The Thermo Scientific Pierce Glutathione Agarose effectively purifies different GST-fusion proteins. Three GST-fusion proteins were expressed in E.

coli and extracted with Thermo Scientific B-PER Reagent with Enzymes (Product # 90078). GST-Pak1 and GST-Bid were purified using the spin format, and GST-

ERK was purified by gravity flow. GST-Pak1 and GST-Bid, 2.4mg and 11mg of total protein, respectively, were purified with a 0.2mL Pierce Glutathione Spin Column

(Product # 16106) per kit instructions. For GST-ERK, 11.2mg of total lysate protein in buffer (50mM Tris, 150mM NaCl; pH 8.0) was applied to a 0.5mL bed of Pierce

Glutathione Agarose (Product #16100) in a drip column and eluted with 125mM Tris, 150mM NaCl, 10mM glutathione at pH 8.0. Chromatography fractions were sep-

arated by SDS-PAGE and stained with Thermo Scientific GelCode Blue Stain Reagent (Product # 24590). M = Molecular weight markers; L = Lysate load; FT = Flow-

through; W = Wash; E = Elution.

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch ofﬁce or distributor.

kDa

250

150

100

M L FT

Thermo

Scientific

Healthcare Qiagen Clontech Sigma

E FT E FT E FT E FT E

Vendor

Thermo

Scientiﬁc

Healthcare Qiagen Clontech Sigma

Yield

537μg 562μg 285μg 299μg 410μg

Purity

93% 93% 90% 91% 94%

Figure 7. Thermo Scientific Pierce Glutathione Agarose delivers high yield

and high-purity GST-fusion proteins. E. coli lysate (14.4mg total protein) con-

taining an overexpressed GST-fusion protein was incubated with 50μL GSH

resin from various suppliers and purified per manufacturers’ instructions. The

amount of GST eluted from the resin (yield) was quantified by Thermo Scientific

Coomassie Plus Protein Assay. Purity was assessed by densitometry of the

stained gel lanes. M= Molecular weight markers; L= Lysate load; FT= Flow-

through; E= Elution.

0.0 10.0 20.0 30.0 40.0 50.0 ml

mAU

4000

Flow-

through

Wash Elution

3000

2000

1000

Figure 8. The glutathione chromatography cartridges provide an automatable

solution for GST-fusion protein purification. Clarified E. coli lysate (50mg)

containing overexpressed GST-Pak1 (34kDa) was applied to a 1mL glutathione

chromatography cartridge in buffer (50mM Tris, 150mM sodium chloride;

pH 8.0) at a flow rate of 0.5mL/minute using an ÄKTApurifier

System. The

cartridge was washed with the same buffer at 1mL/minute. GST-Pak1 was

eluted (50mM Tris, 150mM sodium chloride, 10mM glutathione; pH 8.0) at

1mL/minute.

Ordering Information

See catalog or www.thermoscientiﬁc.com/pierce for a complete description

of products and kits.

Product # Description Pkg. Size

U.S.

Price

16100 Pierce Glutathione Agarose

10mL

$ 180

16101 Pierce Glutathione Agarose

100mL

$ 975

16102 Pierce Glutathione Agarose

500mL

$3000

16103 Pierce Glutathione Spin Columns, 0.2mL

25 columns

$ 175

16104 Pierce Glutathione Spin Columns, 1mL

5 columns

$ 175

16105 Pierce Glutathione Spin Columns, 3mL

5 columns

$ 315

16106 Pierce GST Spin Puriﬁcation Kit, 0.2mL

25-column kit

$ 275

16107 Pierce GST Spin Puriﬁcation Kit, 1mL

5-column kit

$ 275

16108 Pierce GST Spin Puriﬁcation Kit, 3mL

5-column kit

$ 375

16109 Pierce Glutathione Chromatography

Cartridges, 1mL

5 cartridges

$ 230

16110 Pierce Glutathione Chromatography

Cartridges, 5mL

2 cartridges

$ 400

For more information, or to download product instructions, visit www.thermoscientiﬁc.com/pierce

GST- and PolyHis-Tagged Pull-Down Assay Kits

Prepare to discover a new protein:protein interaction with your

GST- or polyHis-tagged bait protein.

Identifying and characterizing the interactions of a given protein has

emerged as the most valuable information that can be developed

in the post-genomic era. Thermo Scientiﬁc Pierce Pull-Down Kits

contain the necessary components to capture interacting proteins

using the popular pull-down method. The only item you provide is an

appropriately tagged fusion protein as the “bait.” Our Pull-Down Kits

are designed to teach the method to the ﬁrst-time user and to shorten

the time to a result for those experienced in this method.

Step 3. Bind “prey” protein to immobilized

“bait” protein.

Step 4. Wash away unbound protein. Step 5. Elute protein:protein interaction complex.

Step 2. Wash away unbound protein.Step 1. Immobilize the fusion-tagged “bait”

from the lysate.

Step 6. Analyze protein:protein interaction

complex on SDS-PAGE.

Fusion Tag (GST or polyHis)

Bait

Prey

Marker Purified

Protein:Protein

Interaction

Agarose

Resin

Control

Purified

Bait

Displaced Interacting Complex

P re y

P ro te in

Bait Protein

Prey

Protein

Bait Protein

Prey Protein-

Containing Lysate

Bait Protein

Bait Protein-

Containing Lysate

Affinity Ligand

(Glutathione or

Chelate)

Agarose

Bead

Prey

Protein

Bait Protein

Prey

Protein

Bait Protein

Prey

Protein

Glutathione

Imidazole

Spin

Highlights:

• Provides a complete, affordable and easy-to-use strategy

for discovery of protein:protein interactions

• Uses common laboratory equipment (e.g., microcentrifuges

and mini-gels)

• Adapts to single- or multiple-sample demands

• Supplied complete with cell lysis buffer

• Flexible protocol aids in the capture of weak or transient

interactions

• Efﬁcient recovery of interacting complexes

Applications:

• Discover a new protein:protein interaction from a cell lysate

• Conﬁrm a putative interaction from a cell lysate or with a

previously puriﬁed protein

• Extract protein:protein interaction information from in vitro

transcription/translation lysates

Generalized scheme for use of a Thermo Scientiﬁc Pierce Pull-Down Protein:Protein Interaction Kit using a GST-tagged or

PolyHis-tagged protein as the “bait.”

Fusion Protein Puriﬁcation

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch ofﬁce or distributor.

Ordering Information

Product #

Description

Pkg. Size

U.S.

Price

21516 Pierce Pull-Down GST Protein Interaction Kit

Sufﬁcient materials for performing 25 pull-down

assays using a GST-tagged protein as the bait.

Includes: Immobilized Glutathione

Lysis Buffer

Glutathione

BupH Tris Buffered Saline

Spin Columns Accessory Pack

Collection Tubes and Caps

Accessory Pack

Kit

750μL settled gel

250mL

1 pack (500μL)

27 columns

100 tubes

$320

Acknowledgment

We gratefully acknowledge Dr. Yigong Shi of Princeton University for supplying the

GST-BIR2- and 9xHis Smac/DIABlO-expressing clones. Dr. Shi’s laboratory is engaged in

research aimed at understanding the structural and molecular mechanisms involved in

tumorigenesis and apoptosis.

Ordering Information

Product #

Description

Pkg. Size

U.S.

Price

21277 Pierce Pull-Down PolyHis

Protein Interaction Kit

Sufﬁcient materials for performing 25 pull-down

assays using a polyhistidine-tagged protein as the bait.

Includes: Immobilized Glutathione

Lysis Buffer

4 M Imidazole Stock Solution

BupH Tris Buffered Saline

Spin Columns Accessory Pack

Collection Tubes and Caps

Accessory Pack

Kit

750μL settled gel

250mL

5mL

1 pack (500μL)

27 columns

100 tubes

$320

References

1. Chai, J., et al. (2000). Nature 406, 855-862.

2. Kaelin, W.G., et al. (1991). Cell 64, 521-532.

3. Sambrook, J. and Russell, D.W. (2001). Molecular Cloning: A Laboratory Manual

3rd Edition. Chapter 18: Protein Interaction Technologies, Protocol #3: Detection of

Protein-Protein Interactions using the GST Fusion Protein Pull-Down Technique.

Cold Spring Harbor Laboratory Press.

4. Soutoglou, E., et al. (2000). Mol. Cell 5, 745-751.

Lane # A. GST-Tag Pull-Down B. PolyHis-Tag Pull-Down

Lysate from E. coli expressing GST-tagged BIR2 (bait protein). Lysate from E. coli expressing 9xHis-tagged wild-type Smac (bait protein).

Flow-through from the lysate in Lane 1 bound to an immobilized

reduced glutathione support for 1 hour at 4°C.

Flow-through from the lysate in Lane 1 bound to an immobilized cobalt chelate support

for 1 hour at 4°C.

Wash #1 of the support. Wash #1 of the support.

Wash #2 of the support. (Washes 3-5 not shown.) Wash #2 of the support. (Washes 3-5 not shown.)

Lysate from E. coli expressing 9xHis-tagged wild-type Smac

(prey protein).

Lysate from E. coli expressing GST-tagged BIR2 (prey protein).

Flow-through from the lysate in Lane 5 is allowed to interact

with the immobilized bait for 1 hour at 4°C.

Flow-through from the lysate in Lane 5 is allowed to interact with the immobilized bait

for 1 hour at 4°C.

Wash #1 of the support. Wash #1 of the support.

Wash #2 of the support. (Washes 3-5 not shown.) Wash #2 of the support. (Washes 3-5 not shown.)

Bait control. Bait treated as described in Lanes 1-8 and

subsequently eluted. No prey added – just binding buffer.

Bait control. Bait treated as described in Lanes 1-8 and subsequently eluted. No prey

added – just binding buffer.

Prey control. Prey treated as described in Lanes 1-8 and

subsequently eluted. No bait added – just binding buffer.

Prey control. Prey treated as described in Lanes 1-8 and subsequently eluted. No bait

added – just binding buffer.

Elution of bait:prey complex (prepared in Lanes 1-8) from the

support with 100mm reduced glutathione. Western blotting

conﬁrms that the minor bands observed in Lanes 9 and 11

are degradation products of GST-tagged BIR2.

Elution of bait:prey complex (prepared in Lanes 1-8) from the support with 250mm imidazole.

Validation of the Thermo Scientiﬁc Pierce Pull-Down Protein Interaction Kits using a known interacting pair.

1 2 3 4 5 6 7 8 9 10 11

A. B.

For more information, or to download product instructions, visit www.thermoscientiﬁc.com/pierce

Covalent Immobilization of Ligands

Afﬁnity chromatography uses the speciﬁc interactions between two

molecules for the puriﬁcation of a target molecule. In practice, a

ligand having afﬁnity for a target molecule is covalently attached to

an insoluble support and functions as bait for capturing the target

from complex solutions.

The afﬁnity ligand can be virtually any molecule that can speciﬁcally

bind the target without displaying signiﬁcant nonspeciﬁc binding

toward other molecules in the solution. Ligands that have been

used for afﬁnity separations include small organic compounds that

are able to dock into binding sites on proteins, inorganic metals that

form coordination complexes with certain amino acids in proteins,

hydrophobic molecules that can bind nonpolar pockets in biomole-

cules, proteins with speciﬁc binding regions that are able to interact

with other proteins, and antibodies, which can be designed to target

any biomolecule through their antigen-binding sites.

The concept of using immobilized afﬁnity ligands to target

biomolecules has extended beyond chromatographic applications.

Afﬁnity ligands are now coupled to latex beads, nanoparticles,

macro-beads, membranes, microplates, array surfaces, dipsticks

and a host of other devices that facilitate the capture of speciﬁc

biomolecules. The application of afﬁnity targeting includes

puriﬁcation, scavenging (or removal of contaminants), catalysis

(or modiﬁcation of target molecules) and a broad range of

analytical uses to quantify a target molecule in a sample solution.

Designing custom afﬁnity supports that are able to target unique

biomolecules requires methods to covalently link a ligand to an

insoluble matrix. Regardless of the intended application, the

chemical reactions that make ligand attachment possible are

well characterized and facilitate the attachment of biomolecules

through their common chemical groups. The types of functionalities

generally used for attachment include easily reactive components

such as primary amines, sulfhydryls, aldehydes, carboxylic acids,

hydroxyls, phenolic groups and histidinyl residues. Usually, the

solid-phase matrix ﬁrst is activated with a compound that is

reactive toward one or more of these functional groups. The

activated complex then can form a covalent linkage between

the ligand and the support, resulting in ligand immobilization.

The type of linkage that is formed between the matrix and the

immobilized ligand affects the performance of the afﬁnity support

in a number of ways. A linkage that allows the coupled ligand to

leach from the matrix will result in contamination of the puriﬁed

protein and shorten the useful life of the afﬁnity support. A linkage

that introduces a charged functional group into the support can

cause nonspeciﬁc binding by promoting ion-exchange effects.

A linkage that alters the structure of the matrix can change the

ﬂow and binding characteristics of the support. Cyanogen

bromide (CNBr)-activated supports are informative as an example

of these principles. This popular immobilization method results

in a linkage that:

1. Has a constant leakage of ligand from the matrix that

becomes a contaminant in the puriﬁed preparation.

2. Includes a charged isourea group in the linkage,

resulting in nonspeciﬁc binding.

3. Causes extensive crosslinking of the matrix, reducing the

ability of large molecules to penetrate into the interior of

the resin.

We offer a number of activated afﬁnity supports that are designed

to couple ligands of every type via stable, uncharged covalent

linkages that avoid introducing undesirable properties into the

supports. The activation chemistry and protocols have been

optimized to ensure excellent coupling yields with minimal effort

under a variety of conditions. Each activated support comes with

instructions for use and literature references as examples. The

associated kits contain all the coupling buffers, wash buffers and

columns necessary to perform the ligand immobilization and

produce a support ready to perform an afﬁnity separation.

Covalent Coupling of Afﬁnity Ligands

to Chromatography Supports

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch ofﬁce or distributor.

Coupling Afﬁnity Ligands through Amine Groups

The most common functional target for immobilizing protein

molecules is the amine group, which is present on the vast

majority of proteins because of the abundance of lysine side chain

e-amines and N-terminal a-amines. Thermo Scientiﬁc AminoLink

Coupling Resin and AminoLink Plus Coupling Resin are prepared

from crosslinked agarose supports, and they are designed to

create a stable linkage between amine groups and the support

material. AminoLink

Resins are activated to contain numerous

aldehyde groups, which can be used to immobilize amine-containing

ligands by reductive amination.

The immobilization reaction using reductive amination involves

the formation of an initial Schiff base between the aldehyde and

amine groups, which then is reduced to a secondary amine by

the addition of sodium cyanoborohydride. The cyanoborohydride

reducing agent used during the coupling process is mild enough

not to cleave disulﬁdes in most proteins, and it will not reduce the

aldehyde reactants – only the Schiff base intermediates. It is best

to avoid stronger reducing agents such as sodium borohydride

because of the potential for disulﬁde reduction of the protein and

reduction of the aldehydes on the support to hydroxyls, effectively

quenching the reaction. Depending on the type and amount of

ligand present, a coupling reaction using reductive amination can

achieve immobilization yields of greater than 85%.

N–R

NaCNBH

Aldehyde Group on

Thermo Scientific

AminoLink Coupling Resin

Affinity Ligand Coupled

via Secondary Amine Bond

Another amine-reactive strategy that can be used for immobilization

is the azlactone ring present in UltraLink Biosupport. A primary

amine will react with an azlactone group in a ring-opening

process that produces an amide bond at the end of a ﬁve-atom

spacer. The group is spontaneously reactive with amines,

requiring no additives or catalysts to drive the coupling process.

The UltraLink Biosupport is supplied dry to ensure stability of the

azlactone groups until use. Adding a quantity of the support to a

sample containing a protein or other amine-containing molecule

causes immobilization to occur within about one hour. For protein

immobilization at high yield, it is recommended that the coupling

buffer contain a lyotropic salt, which functions to drive the protein

molecules toward the bead surface. This brings the hydrophilic

amines close enough to the azlactone rings to react quickly. The

simple nature of coupling afﬁnity ligands to the UltraLink

Biosupport along with its inherently low nonspeciﬁc binding makes

it one of the best choices for immobilization.

Azlactone Group on

Thermo Scientific

UltraLink Biosupport

Affinity Ligand Coupled

via Amide Bond

N—R

Thermo Scientiﬁc UltraLink Biosupport binding capacity for various proteins.

Capacity Protein Coupling Buffer

35.0mg/mL Myoglobin 0.1M CHES, 1.0M sodium citrate, pH 9.0

21.5mg/mL Penicillin Acylase 0.1M sodium phosphate, 1.1M sodium

sulfate, pH 7.4

20.9mg/mL a-chymotrypsin 0.1M borate, 1.5 sodium sulfate, pH 9.0

35.5mg/mL BSA 0.1M borate, 1.5 sodium sulfate, pH 9.0

29.8mg/mL Lysozyme 0.1M borate, 1.0 sodium sulfate, pH 9.0

21.0mg/mL Human IgG 0.1M borate, 1.5 sodium sulfate, pH 9.0

A third option for immobilizing amine-containing afﬁnity ligands

is the use of carbonyl diimidazole (CDI) to activate hydroxyls on

agarose supports to form reactive imidazole carbamates. This

reactive group is formed on the support in organic solvent and

stored as a suspension in acetone to prevent hydrolysis. Reaction

of the support in an aqueous coupling buffer with primary

amine-containing ligands causes loss of the imidazole groups

and formation of carbamate linkages. The coupling process

occurs at basic pH (8.5–10), but it is a slower reaction with

proteins than reductive amination or azlactone coupling. Thermo

Scientiﬁc Pierce CDI Supports are available with the CDI-activated

group, and they are particularly adept at immobilizing peptides and

small organic molecules. The reaction also can be done in

organic solvent to permit coupling of water-insoluble ligands.

Reactive Imidazole Carbamate

on Thermo Scientific Pierce

CDI Supports

Affinity Ligand Coupled

via Carbamate Bond

N—R

NHS-Activated Agarose resin uses safe, reliable NHS-ester

chemistry and does not require hazardous chemicals for

immobilization. Other amine-reactive supports, such as periodate-

oxidized resins, use toxic sodium cyanoborohydride to stabilize

the reaction linkage to primary amines and take 4 to 6 hours

to complete. Traditional methods such as cyanogen bromide-

activated supports also couple amines; however, this chemistry

results in nonspeciﬁc binding and constant slow leakage of the

coupled ligand. The Pierce NHS-Activated Agarose uses the

stable NHS-ester chemistry that allows immobilization reactions

to be completed in less than 1 hour.

The NHS-Activated Agarose coupling reaction is performed in an

amine-free buffer at pH 7-9. Protein coupling efﬁciency is typically

greater than 80%, regardless of the ligand’s molecular weight or pI.

Once a ligand is immobilized, the prepared resin can be used for

multiple afﬁnity puriﬁcation procedures. The crosslinked beaded

agarose has fast linear ﬂow potential, making it useful for gravity-

ﬂow and low- to medium-pressure applications.

For more information, or to download product instructions, visit www.thermoscientiﬁc.com/pierce

NHS-Activated Agarose resin is available in an anhydrous acetone

slurry and as dry powder. The unique dry form does not require

storage in or removal and disposal of the acetone solvent. In

addition, the dry resin is ideal for coupling reactions with dilute

samples because it concentrates the sample as the resin swells,

reducing the volume of the starting material and resulting in highly

efﬁcient ligand immobilization.

–R

Coupling Afﬁnity Ligands through Sulfhydryl Groups

It is often advantageous to immobilize afﬁnity ligands through

functional groups other than just amines. In particular, the thiol

group can be used to direct coupling reactions away from active

centers or binding sites on certain protein molecules. Because

amines occur at many positions on a protein’s surface, it is usually

difﬁcult to predict where an amine-targeted coupling reaction will

occur. However, if sulfhydryl groups that typically are present in

fewer numbers are targeted for immobilization, then coupling can

be done at discrete sites in a protein or peptide. Thiol groups

(sulfhydryls) may be indigenous within a protein molecule or they

can be added through the reduction of disulﬁdes or through the

use of various thiolation reagents. Sulfhydryls also can be added

to peptide afﬁnity ligands at the time of peptide synthesis by

adding a cysteine residue at one end of the molecule. This ensures

that every peptide molecule will be oriented on the support in the

same way after immobilization.

Thermo Scientiﬁc SulfoLink Coupling Resin is designed to

efﬁciently react with thiol-containing molecules and immobilize

them through a thioether linkage. The support contains an iodo-

acetyl group at the end of a long spacer arm, which reacts with

sulfhydryls through displacement of the iodine. Optimal conditions

for the reaction are an aqueous environment at slightly basic pH,

wherein amines are not very reactive toward the iodoacetyl

function, but thiols are highly reactive due to their increased

nucleophilicity. The thioether bond that is formed provides a

stable linkage to any sulfhydryl-containing molecule.

Reactive Iodoacetyl Group

on Thermo Scientific

SulfoLink Supports

Affinity Ligand

Coupled via

Thioether Bond

Coupling Afﬁnity Ligands through Carbonyl Groups

Most biological molecules do not contain carbonyl ketones or

aldehydes in their native state. However, it might be useful to

create such groups on proteins to form a site for immobilization that

directs covalent coupling away from active centers or binding sites.

Glycoconjugates, such as glycoproteins or glycolipids, usually

contain sugar residues that have hydroxyls on adjacent carbon

atoms, which can be periodate-oxidized to create aldehydes.

Controlled oxidation using 1mm sodium meta-periodate at 0°C will

selectively oxidize sialic acid groups to form an aldehyde functionality

on each sugar. Using higher concentrations of periodate (10mm)

at room temperature will result in oxidation of other sugar diols to

create additional formyl groups. Aldehydes on the carbohydrate

portion of glycoconjugates can be covalently linked with

afﬁnity supports through an immobilized hydrazide, hydrazine or

amine group by Schiff base formation or reductive amination.

Thermo Scientiﬁc CarboLink Coupling Resin contains long spacer

arms that terminate in hydrazide groups. Reaction of the hydrazides

with aldehydes forms hydrazone linkages, which are a form of

Schiff base displaying better stability than those formed between

an amine and an aldehyde. The CarboLink

™

Resin can be used to

immobilize glycoproteins, such as antibodies, after periodate

oxidation of the carbohydrate. Coupling antibodies in this manner

speciﬁcally targets the heavy chains in the Fc portion of the

molecule. Since this is away from the antigen-binding sites at the

end of the Fv regions, immobilization using this route often results

in the best retention of antigen-binding activity.

Reactive Hydrazide Group

on Thermo Scientific

CarboLink Supports

Affinity Ligand

Coupled via

Hydrazone Bond

R H

Covalent Coupling of Afﬁnity Ligands

to Chromatography Supports

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch ofﬁce or distributor.

The CarboLink Resin also can be used to couple carbohydrates and

sugars through their reducing ends. Aldehyde- or ketone-containing

sugars will react with the immobilized hydrazide groups without

oxidation of other sugar hydroxyls. However, this reaction may be

dramatically slower than coupling with oxidized sugars because

these native aldehydes or ketones are usually tied up in acetal or

ketal ring structures. These rings can open in aqueous solution to

reveal the aldehyde or ketone, but the open structure is present

only a small percentage of the time. Thus, the reducing ends of

sugars have decreased reactivity toward an immobilized hydrazide,

sometimes requiring days of reaction time to obtain acceptable

immobilization yields.

Although the hydrazone bond created between the immobilized

hydrazide and an aldehyde is much more stable than amine-aldehyde

Schiff bases, to obtain a leach-resistant linkage it is recommended

that the Schiff base be reduced with sodium cyanoborohydride.

This is especially true if a ligand is coupled that has only a single

point of attachment to the support. Reduction of the hydrazone

creates a stable bond that will perform well in afﬁnity

chromatography applications.

Affinity Ligand

Coupled via

Hydrazone Bond

Stable Ligand

Linkage

NaCNBH

Coupling Afﬁnity Ligands through Carboxyl Groups

The carboxyl group is a frequent constituent of many biological

molecules. Particularly, proteins and peptides typically contain

numerous carboxylic acids due to the presence of glutamic acid,

aspartic acid and the C-terminal a-carboxylate group. Carboxylic

acids may be used to immobilize biological molecules through the

use of a carbodiimide-mediated reaction. Although no activated

support contains a reactive group that is spontaneously reactive

with carboxylates, chromatography supports containing amines

(or hydrazides) may be used to form amide bonds with carboxyl-

ates. Molecules containing carboxylates may be activated to react

with an immobilized amine (or hydrazide) through reaction with the

water-soluble carbodiimide EDC.

EDC reacts with carboxylates to form an intermediate ester that is

reactive with nucleophiles such as primary amines. The reaction

takes place efﬁciently between about pH 4.5 and pH 7.5, and it is

complete within two to four hours, depending on the temperature.

The intermediate ester is subject to hydrolysis; therefore, it is

beneﬁcial if the amine-containing ligand to be immobilized is

included in the reaction medium upon addition of EDC, so it can

react immediately with the ester as it forms.

Thermo Scientiﬁc CarboxyLink Coupling Resin or the UltraLink

DADPA Resin may be used to immobilize carboxylate-containing

ligands by EDC. CarboxyLink

Resin contains a nine atom spacer

arm and UltraLink DADPA contains a 12-atom spacer arm to mini-

mize steric hindrance.

Carboxylate-Containing

Affinity Ligand

EDC

Immobilized DADPA on

Thermo Scientific CarboxyLink Resin

Immobilized Ligand via

Amide Bond Formation

For more information, or to download product instructions, visit www.thermoscientiﬁc.com/pierce

Products for Immobilizing Ligands through

Primary Amines

Thermo Scientiﬁc Pierce NHS Ester-Activated Agarose

Pierce NHS-Activated Agarose Resin, available as a slurry or

dry powder, allows for the simple and efﬁcient immobilization of

proteins to a beaded-agarose support. The activated agarose

contains N-hydroxysuccinimide (NHS) ester with a spacer arm of

at least 10 atoms in length that reacts with primary amines form-

ing stable amide linkages. The NHS-Activated Agarose coupling

reaction is performed in an amine-free buffer at pH 7-9, with typi-

cal coupling efﬁciencies of more than 85%. The prepared resin

can be used for multiple afﬁnity puriﬁcation procedures. The

crosslinked beaded agarose has fast linear ﬂow potential, making

it useful for gravity-ﬂow and low- to medium-pressure applications.

Agarose

Bead

Agarose

Bead

Protein

Thermo Scientiﬁc NHS Ester-Activated Agarose Support

immobilization chemistry.

Target: –NH

Support: 6% agarose

Binding capacity: >25mg protein/mL resin

Time: 30 minutes

Ordering Information

Product #

Description

Pkg. Size

U.S.

Price

26196

Pierce NHS-Activated Agarose, Dry

Swell volume: 6-7.5mL/g of dry resin

$ 65

26197

Pierce NHS-Activated Agarose, Dry

Swell volume: 6-7.5mL/g of dry resin

$200

26198

Pierce NHS-Activated Agarose

Spin Columns, 0.2mL

25 spin columns containing 33mg

of NHS-Activated Agarose

Swell volume: 6- 7.5uL/mg of dry resin

25 columns

$125

26199

Pierce NHS-Activated Agarose

Spin Columns, 2mL

5 spin columns containing 330mg of

NHS-Activated Agarose

Swell volume: 6- 7.5uL/mg of dry resin

5 columns

$125

26200

Pierce NHS-Activated Agarose Slurry

25mL of settled resin in anhydrous acetone

25mL

$195

Covalent Coupling of Afﬁnity Ligands

to Chromatography Supports

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch ofﬁce or distributor.

Thermo Scientiﬁc AminoLink Plus Immobilization Kits

and Coupling Resin

The simplest and surest method for making an afﬁnity puriﬁcation

resin with antibodies or other proteins.

AminoLink Plus Coupling Resin and Immobilization Kits use activated

beaded agarose and a robust coupling chemistry to immobilize

proteins and other ligands through primary amines (–NH

) to the

resin. Once an antibody or other ligand is immobilized, the prepared

afﬁnity resin can be used for a variety of puriﬁcation methods

involving batch or column chromatography. The resin and linkage

are stable in binding and elution conditions typically used in afﬁnity

chromatography, enabling prepared resin to be used for at least

10 rounds of afﬁnity puriﬁcation.

The AminoLink Plus Coupling Reaction involves spontaneous

formation of Schiff base bonds between aldehydes (on the support)

and amines (on the ligand) and their subsequent stabilization by

incubation with a mild reductant (sodium cyanoborohydride; see

more detailed reaction scheme on next page). The entire coupling

reaction, called reductive amination, occurs in four to six hours

in simple non-amine buffers such as PBS. Coupling efﬁciency with

antibodies and typical proteins is generally greater than 85%,

resulting in 1 to 20mg of immobilized protein per milliliter of

agarose resin.

Highlights:

• AminoLink Plus Coupling Resin – aldehyde-activated crosslinked

4% beaded agarose

• Ideal for antibodies and other proteins – immobilize molecules

via primary amines (–NH

)

• Flexible coupling conditions – efﬁcient (>85%) coupling over

a wide range of pH (4–10) and buffer conditions (PBS or other

non-amine buffer with or without organic solvent); regular

(PBS, pH 7.2) and enhanced (borate, pH 10) coupling

protocols provided

• Stable, permanent immobilization – coupling reaction results

in stable, leak-resistant secondary amine bond between resin

and ligand

• Better than immobilization to CNBr-activated agarose – bond is

more stable and uncharged, resulting in less nonspeciﬁc binding

in afﬁnity puriﬁcation procedures

• Versatile and reusable – prepared afﬁnity resin is adaptable to

column and batch afﬁnity techniques and the resin is reusable

for typical applications based on protein binding interactions

• Convenient kits and product sizes – choose one- or ﬁve-column

kit containing complete sets of buffers, reagents and versatile

spin/drip columns, or select bulk resin. Bulk quantities are

available for manufacturing applications

Efﬁcient immobilization of antibodies and other proteins. Percent coupling

efﬁciency of different proteins to 2mL of Thermo Scientiﬁc AminoLink Plus

Coupling Resin using the pH 7.2 coupling protocol.

Protein

Protein Applied

(mg/mL)

Protein Coupled

(mg/mL)

Percent Coupled

Protein G 4.6 4.0 83

Mouse IgG 4.7 4.5 96

Rat IgG 4.7 4.4 93

Goat IgG 0.9 0.8 84

Human IgG 4.8 4.6 97

Human IgM 0.9 0.8 93

Agarose

Bead

Protein

Agarose

Bead

Protein

Thermo Scientiﬁc AminoLink Support immobilization chemistry.

Target: –NH

Support: Highly crosslinked 4% agarose

Binding capacity: 20mg protein/mL resin

Time: 4 hours

References

Beall, A., et al. (1999). J. Biol. Chem. 274(16), 11344–11351.

Nakasato, Y.R., et al. (1999). Clin. Chem. 45, 2150–2157.

Allan, B.B., et al. (2000). Science 289, 444–448.

Lu, R., et al. (2000). J. Neurochem. 74, 320–326

Ordering Information

Product #

Description

Pkg. Size

U.S.

Price

44894 AminoLink Plus Immobilization Kit

Sufﬁcient reagents for preparing ﬁve afﬁnity columns.

Includes: AminoLink Column

Phosphate Buffered Saline (PBS)

Citrate-Carbonate Buffer

Quenching Buffer

Wash Solution

Sodium Cyanoborohydride Solution (5 M)

Column Accessories

Kit

5 x 2mL

1 pack

50mL

240mL

0.5mL

$385

20394 AminoLink Plus Immobilization Trial Kit

Sufﬁcient reagents for preparing one afﬁnity column.

Includes: AminoLink Column

Phosphate Buffered Saline (PBS)

Citrate-Carbonate Buffer

Quenching Buffer

Wash Solution

Sodium Cyanoborohydride Solution (5 M)

Column Accessories

Kit

1 x 2mL

1 pack

15mL

50mL

0.5mL

$105

20475 AminoLink Plus Micro Immobilization Kit

Sufﬁcient reagents for 10 coupling reactions

using 25–100µg of protein and 20 afﬁnity puriﬁcations.

Includes: AminoLink Plus Spin Columns,

each containing 400μL of 25% slurry

Phosphate Buffered Saline

Quenching Buffer

Sodium Cyanoborohydride Solution

Wash Solution

Elution Buffer

Microcentrifuge Collection Tubes

Kit

10 each

1 pack

60mL

0.5mL

25mL

50mL

200 each

$301

20501 AminoLink Plus Coupling Resin

10mL

$105

20505 AminoLink Plus Coupling Resin

50mL

$360

For more information, or to download product instructions, visit www.thermoscientiﬁc.com/pierce

Thermo Scientiﬁc AminoLink Immobilization Kits

and Coupling Resin

Links with primary amines (lysine residues and N-terminus)

on proteins, peptides, antigens or antibodies.

AminoLink Coupling Resin is crosslinked 4% beaded agarose that

has been activated with aldehyde groups. Proteins and other

molecules with primary amines can be covalently attached

(immobilized) to AminoLink Resin to make chromatography columns

for use in afﬁnity puriﬁcation. The aldehyde groups form stable

secondary amine bonds with primary amines such as exist in the

side chain of lysine (K) residues, which are generally abundant

and readily accessible in proteins. Once a protein is immobilized,

the prepared afﬁnity resin can be used for a variety of batch and

column afﬁnity puriﬁcation methods involving binding interactions

with the immobilized protein. The resin and linkage are stable

in most binding and elution conditions typically used in afﬁnity

chromatography, enabling prepared resin to be used for multiple

rounds of afﬁnity puriﬁcation procedures.

Highlights:

• AminoLink Coupling Resin – aldehyde-activated crosslinked

4% beaded agarose

• Ideal for antibodies and other proteins – immobilize molecules

via primary amines (–NH

)

• Flexible coupling conditions – efﬁcient (>85%) coupling over a

wide range of pH (4–10) and buffer conditions (PBS or other

non-amine buffer with or without organic solvent)

• Stable, permanent immobilization – coupling reaction results in

stable, leak-resistant secondary amine bond between resin

and ligand

• Better than immobilization to CNBr-activated agarose – bond is

more stable and uncharged, resulting in less nonspeciﬁc binding

in afﬁnity puriﬁcation procedures

• Versatile and reusable – prepared afﬁnity resin is adaptable to

column and batch afﬁnity techniques and the resin is reusable

for typical applications based on protein binding interactions

Agarose

Bead

Protein

Agarose

Bead

Protein

Thermo Scientiﬁc AminoLink Support detailed immobilization chemistry.

Target: –NH

Support: 4% agarose

Binding capacity: 1-20mg protein/mL resin

Time: 4 hours

Efﬁcient immobilization at a variety of pH values. The effect of coupling

buffer pH on percent coupling efﬁciency of 9.58mg of human IgG to 2mL of

Thermo Scientiﬁc AminoLink Resin using the standard coupling protocol.

pH Coupling Efﬁciency of 9.58mg Human IgG

4 91.8%

5 92.7%

6 89.1%

7 87.3%

8* 85.3%

9* 94.9%

10* 98.4%

* Schiff-base formation occurs readily at high pH, but reduction to stable secondary

amine bond requires subsequent incubation with sodium cyanoborohydride

(AminoLink Reductant) at pH 7.2.

References

Cheadle, C., et al. (1994). J. Biol. Chem. 269(39), 24034–24039.

Cofano, F., et al. (1990). J. Biol. Chem. 265(7), 4064–4071.

DeSilva, B.S. and Wilson, G.S. (1995). J. Immunol. Method 188, 9–19.

Rivero-Lezcano, O.M., et al. (1994). J. Biol. Chem. 269(26), 17363–17366.

Czermak, B.J., et al. (1999). J. Immunol. 162, 2321–2325.

Assad, F.F., et al. (2001). J. Cell Biol. 152, 531–543.

Zuk, P.A. and Elferink, L.A. (2000). J. Biol. Chem. 275(35), 26754–26764.6.

Ordering Information

Product #

Description

Pkg. Size

U.S.

Price

20381 AminoLink Coupling Resin

10mL

$105

20382 AminoLink Coupling Resin

50mL

$380

44890 AminoLink Immobilization Kit

Sufﬁcient reagents to prepare ﬁve afﬁnity columns.

Each column can be used for up to

10 afﬁnity puriﬁcations.

Includes: AminoLink Columns

AminoLink Coupling Buffer

Quenching Buffer

Wash Solution, Sodium Cyanoborohydride

Solution (5 M)

Column Accessories

Kit

5 x 2mL

250mL

50mL

240mL

0.5mL

$399

20384 AminoLink Immobilization Trial Kit

Includes: AminoLink Column Reagents and Buffers

Trial Kit

1 x 2mL

$106

44892 AminoLink Reductant

(Sodium cyanoborohydride)

2 x 1g

$ 39

Covalent Coupling of Afﬁnity Ligands

to Chromatography Supports

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch ofﬁce or distributor.

Thermo Scientiﬁc UltraLink Biosupport

A durable, polyacrylamide resin, activated for efﬁcient coupling

of proteins.

UltraLink Biosupport is a durable, porous resin that is activated to

enable efﬁcient and direct covalent immobilization of proteins and

other biomolecules through their primary amines for use in afﬁnity

puriﬁcation procedures.

Highlights:

• High coupling efﬁciency and capacity – immobilizes proteins

with very high efﬁciency and coupling capacity in 1 hour

• Speciﬁc and leak-proof coupling chemistry – reacts speciﬁcally

with primary amines (–NH

), resulting in amide bonds that are

stable for use in many afﬁnity puriﬁcation procedures; coupling

reaction has no leaving group to contaminate samples

• Easy to use – no pre-swelling or secondary reagents required;

simply weigh the needed amount of dry support and add the

ligand solution to initiate coupling reaction

• Flexible coupling conditions – perform immobilization reaction

in any of a variety of non-amine buffers and pH levels; coupling

is most efﬁcient in buffers containing a lyotropic salt such as

sodium citrate; coupling compatible with or without organic solvent

• Excellent reusability – prepared afﬁnity resin can be used with

typical binding and elution procedures for more than 100 cycles

of afﬁnity puriﬁcation without signiﬁcant loss of binding capacity

• Durable, high-performance resin – porous beads have a 60μm

diameter, can withstand 100 psi (6.9 bar) and allow for linear

ﬂow-through rates of 3,000cm/hour

Amine

Ligand

Protein

UtraLink Biosupport

(Azlactone Ring on Bead)

Amide Bond Formation

with Ring Opening

Resin

Bead

Resin

Bead

Protein

Thermo Scientiﬁc UltraLink Biosupport immobilization chemistry.

Target: –NH

Support: Polyacrylamide/azlactone copolymer

Binding capacity: >18mg protein/mL resin

Time: 4 hours

References

1. Ju, T., et al. (2002). J. Biol. Chem. 277, 169–177.

2. Ju, T., et al. (2002). J. Biol. Chem. 277, 178–186.

3. Kornfeld, R., et al. (1998). J. Biol. Chem. 273, 23202–23210.

4. Liu, L.A. and Engvall, E. (1999). J. Biol. Chem. 274, 38171–38176.

Ordering Information

Product #

Description

Pkg. Size

U.S.

Price

53110 UltraLink Biosupport (8-10mL)

1.25g

$155

53111 UltraLink Biosupport (50mL)

6.25g

$539

28388 BupH Citrate-Carbonate Buffer Packs

10 packs

$ 64

28386 BupH Citrate-MOPS Buffer Packs

10 packs

$ 58

Thermo Scientiﬁc Pierce CDI Supports

Carbonyldiimidazole-activated resins for ligand immobilization.

Highlights:

• Reliable coupling chemistry – immobilization occurs through the

reaction of N-nucleophiles with 1,1’-carbonyldiimidazole groups

of the resin to form a stable, uncharged N-alkylcarbamate linkages

• Easy-to-use – no secondary reagents needed; just wash equilibrate

resin in alkaline coupling buffer and add ligand; reaction proceeds

spontaneously

• Stable activation – half-life of hydrolysis is much longer than

hydroxysuccinimide ester activations, making immobilization

reactions simpler to prepare and control; simpliﬁes ﬁltration and

washing before adding a ligand or protein

• Well-deﬁned coupling conditions – reaction is most efﬁcient with

primary amines at pH 9-11

CDI-Activated Crosslinked 6% Beaded Agarose:

• Beaded agarose – the most popular resin for routine afﬁnity

puriﬁcation methods

• Highly activated – at least 50μmol 1,1’-carbonyldiimidazole (CDI)

groups per milliliter of resin

• Convenient form – supplied as stabilized, 50% slurry in acetone

CDI-Activated Trisacryl

GF-2000:

• Trisacryl resin – rigid, polyacrylamide matrix allows for high ﬂow rates

• Hydrophilic matrix without charge effects – provides for low

nonspeciﬁc binding

• Highly activated – at least 50μmol 1,1’ -carbonyldiimidazole (CDI)

groups per milliliter of resin

• Convenient form – supplied as stabilized, 50% slurry in acetone

Resin

Bead

Imidazole

Carbamate

Amine

Ligand

Carbamate

Linkage

Resin

Bead

Protein

CDI immobilization chemistry.

References

1. Shenoy, S.K., et al. (2001). Science 294, 1307–1313.

2. Richardson, R.T., et al. (2000). J. Biol. Chem. 275, 30378–30386.

3. Tanaka, M, et al. (2005). PLoS Biology 3, 764–776.

Ordering Information

Product #

Description

Pkg. Size

U.S.

Price

20259 Pierce CDI (6X) Support

1,1’-Carbonyldiimidazole activated crosslinked

6% beaded agarose

Supplied: stabilized in acetone slurry agarose

hydrated particle size: 45–165μm

Activation level: >50μmol/mL of resin

10mL

$ 85

20377 Pierce CDI Trisacryl Support

50mL

$442

For more information, or to download product instructions, visit www.thermoscientiﬁc.com/pierce

Products for Immobilizing Ligands

through Sulfhydryl Groups

Thermo Scientiﬁc SulfoLink Immobilization Kits

and Coupling Resin

Covalent immobilization of sulfhydryl-containing peptides or

proteins for afﬁnity puriﬁcation.

SulfoLink Coupling Resin is porous, crosslinked 6% beaded agarose

that has been activated with iodoacetyl groups. When incubated

with a solution of peptide or protein that contains reduced cysteine

residues, the iodoacetyl groups react speciﬁcally and efﬁciently

with the exposed sulfhydryls (–SH) to form covalent and irrevers-

ible thioether bonds that permanently attach the peptide or protein

to the resin. The result is a custom-made afﬁnity resin for puriﬁca-

tion of antibodies, antigens and other molecules of interest.

Highlights:

• Speciﬁc conjugation through sulfhydryl (–SH) groups – the

iodoacetyl groups react speciﬁcally with sulhydryls to form

irreversible thioether bonds

• Separate kits optimized for peptides or proteins – kits include

optimized reagents for preparing peptide or protein samples for

efﬁcient immobilization

• Fast – spin columns increase protocol speed; prepare and

couple samples in 2 hours (peptides) to 3.5 hours (proteins)

• Flexible coupling conditions – use pH 7.5–9.0 aqueous buffers,

organic solvent (e.g., 20% DMSO) or denaturant (guanidine•HCl),

as needed for protein or peptide solubility during coupling reaction

• Easy-to-follow instructions – streamLined protocols for sample

preparation, immobilization, and afﬁnity puriﬁcation

• High capacity – immobilize 1–2mg peptide or 2–20mg protein

per 2–mL column of SulfoLink Coupling Resin

Thermo Scientific

SulfoLink Coupling Resin

Thioether

Bond

12-atom

Spacer

Iodoacetyl

Group

Immobilized Peptide

Reduced Sulfhydryl

Molecule

Agarose

Bead

Peptide

Agarose

Bead

Thermo Scientiﬁc SulfoLink Support immobilization chemistry.

Target: –SH

Support: 6% agarose or UltraLink

Resin

Binding capacity: 20mg protein/mL resin

Time: 2-3.5 hours

Peptide A Calcitonin Peptide

100

+TCEP

-TCEP

Percent

Immobilized

Improved retention of peptides on Thermo Scientiﬁc SulfoLink Resin with TCEP.

TCEP effectively reduces peptides to maximize immobilization efﬁciency. Two

peptides (Peptide A and human calcitonin peptide) were incubated with 25mm

TCEP for 30 minutes and immobilized onto SulfoLink Resin via their reduced

sulfhydryl groups. Peptide A’s cysteine had oxidized during long-term storage and

the calcitonin peptide contained an internal disulﬁde bond. Each peptide also con-

tained an amine-terminal ﬂuorescent probe by which the binding of the peptide

could be monitored during the immobilization and wash steps.

References

Grunwald, R. and Meissner, G. (1995). J. Biol. Chem. 270(19), 11338–11347.

Seubert, P., et al. (1993). Nature 361, 260–263.

Sukegawa, J., et al. (1995). J. Biol. Chem. 270(26), 15702–15706.

Wisniewski, J.R., et al. (1994). J. Biol. Chem. 269(46), 29261–29264.

Sakaguchi, K., et al. (2000). J. Biol. Chem. 275, 9278–9283.

Tan, M., et al. (2000). Proc. Natl. Acad. Sci. USA 97, 109–114.

Quill, T.A., et al. (2001). Proc. Natl. Acad. Sci. USA 98, 12527–12531.

Tokumaru, H., et al. (2001). Cell 104, 421–432.

Assad, F.F., et al. (2001). J. Cell Biol. 152, 531–543.

Ordering Information

Product #

Description

Pkg. Size

U.S.

Price

44995 SulfoLink Immobilization Kit for Proteins

Sufﬁcient reagents to prepare ﬁve afﬁnity columns.

Kit contains: SulfoLink Columns

SulfoLink Sample Preparation Buffer

SulfoLink Coupling Buffer

Wash Solution

2-Mercaptoethylamine•HCl

L-Cysteine•HCl

Zeba Desalt Spin Columns

Phosphate Buffered Saline

Columns Accessories

Kit

5 x 2mL

7.5mL

500mL

120mL

5 x 6mg

100mg

5 x 5mL

1 Pack

$ 399

44999 SulfoLink Immobilization Kit for Peptides

Sufﬁcient reagents to prepare ﬁve afﬁnity columns.

Kit contains: SulfoLink Columns

SulfoLink Coupling Buffer

Wash Solution

Bond-Breaker TCEP Solution

L-Cysteine•HCl

Phosphate Buffered Saline

Column Accessories

Kit

5 x 2mL

120mL

0.5mL

100mg

1 Pack

$ 399

20325 SulfoLink Immobilization Trial Kit

Sufﬁcient reagents to prepare 1 afﬁnity column with a

peptide or protein.

Kit contains: SulfoLink Column

SulfoLink Sample Preparation Buffer

SulfoLink Coupling Buffer

Wash Solution

2-Mercaptoethylamine•HCl

Bond-Breaker TCEP Solution

Cysteine•HCl

Zeba Desalt Spin Column

Phosphate Buffered Saline

Columns Accessories

Kit

1 x 2mL

7.5mL

120mL

25mL

6mg

0.5mL

100mg

5mL

1 Pack

$ 170

20401 SulfoLink Coupling Resin

10mL

$ 135

20402 SulfoLink Coupling Resin

50mL

$ 474

20404 SulfoLink Coupling Resin

250mL

$1900

Covalent Coupling of Afﬁnity Ligands

to Chromatography Supports

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch ofﬁce or distributor.

Thermo Scientiﬁc UltraLink Iodoacetyl Resin

Polyacrylamide resin for coupling sulfhydryl-containing ligands.

UltraLink Iodoacetyl Resin is a durable, porous resin that is activated

to enable efﬁcient and direct covalent immoblization of peptides

and other ligands through their sulfhydryl groups (–SH) for use in

afﬁnity puriﬁcation procedures. The beaded resin is a hydrophilic

copolymer of polyacrylamide and azlactone having a rigid poly-

meric structure with high surface area and pore volume. Each

azlactone group has been modiﬁed to form a 15-atom spacer arm

that terminates in an iodoacetyl group, which is capable of react-

ing with sulfhydryl groups (e.g., side chain of reduced cysteine

residues) to covalently immobilize peptide or other sulfhydryl-

containing ligands. The bead structure and efﬁciently coupling

chemistry of Iodoacetyl-Activated UltraLink Support results in high

protein binding capacity, high linear ﬂow rates, low nonspeciﬁc

binding and overall superior performance in afﬁnity chromatography.

UltraLink Resins are ideal for medium pressure applications such

as FPLC.

Highlights:

• High coupling efﬁciency and capacity – immobilizes sulfhydryl-

containing proteins or other ligands with high efﬁciency and

coupling capacity in 1 hour

•

Speciﬁc and leak-proof coupling chemistry – reacts speciﬁcally

with sulfhydryl groups (reduced thiols), resulting in thioether

bonds that are stable for use in many afﬁnity puriﬁcation procedures

•

Simple coupling conditions – perform immobilization reaction in

any of a variety of buffers; coupling is most efﬁcient and speciﬁc

at pH 8.0–8.5; coupling compatible with or without organic solvent

•

Excellent reusability – prepared afﬁnity resin can be used with

typical binding and elution procedures for many cycles of affnity

puriﬁcation without signiﬁcant loss of binding capacity

•

Durable, high-performance resin – porous beads have a 60μm

diameter, can withstand 100 psi (6.9 bar) and allow for linear

ﬂow-through rates of 3,000cm/hour

References

1. Ju, T., et al. (2002). J. Biol. Chem. 277, 169–177.

2. Hill, K., et al. (2000). J. Biol. Chem. 275(6), 3741–3744.

3. Liu, L.A. and Engvall, E. (1999). J. Biol. Chem. 274, 38171–38176.

4. Bicknell, A.B., et al. (2001). Cell 105, 903–912.

Ordering Information

Product #

Description

Pkg. Size

U.S.

Price

53155

UltraLink Iodoacetyl Resin

Support: UltraLink Biosupport

10mL

$188

Iodoacetyl-Activated

Thermo Scientific UltraLink Resin

Thioether

Bond

15-atom

Spacer

Iodoacetyl

Group

Immobilized Peptide

Reduced Sulfhydryl

Molecule

Thermo Scientific

UltraLink Bead

Thermo Scientific

UltraLink Bead

Peptide

Thermo Scientiﬁc UtraLink Iodoacetyl immobilization chemistry.

Thermo Scientiﬁc UltraLink Iodoacetyl Micro Peptide Coupling Kit

Easily prepare a small-scale afﬁnity column with sulfhydryl-

containing peptides.

The Micro Peptide Coupling Kit is a microcentrifuge spin

column kit for immobilizing small amounts (25–250μg) of sulfhydryl-

containing peptides (e.g., cysteine-terminated peptides) onto a

beaded porous resin to create a small, reusable afﬁnity column.

The coupling and afﬁnity puriﬁcation procedures are optimized

for small sample volumes (200–300μL). Wash and elution steps are

achieved rapidly and efﬁciently with the convenient microcentrifuge

spin columns. Each kit contains sufﬁcient reagents for 10 coupling

reactions and 20 afﬁnity puriﬁcations. The kit is ideal for immobilizing

peptide antigens that containing a terminal cysteine residue for

use in purifying speciﬁc antibodies from small serum, ascites or

culture supernatant samples.

Highlights:

• Optimized for small scale – ideal for coupling small amounts

(25–250μg) of sulfhydryl-containing peptide or protein for

puriﬁcation of speciﬁc antibodies from crude serum

•

High-performance afﬁnity resin – uses durable, polyacrylamide-

based UltraLink Iodoacetyl Resin for speciﬁc reaction to

sulfhydryl groups

•

Efﬁcient coupling chemistry – immobilization efﬁciency >85%

(as measured with 1 hour reaction using insulin, calcitonin and

osteocalcin peptides)

•

Fast and easy to use – perform wash and elution steps using

a microcentrifuge

•

Reusable – use prepared peptide resin several times with no

signiﬁcant loss of capacity

Ordering Information

Product #

Description

Pkg. Size

U.S.

Price

20485

Micro Peptide Coupling Kit

Sufﬁcient for coupling 10 sulfhydryl-containing peptides

or proteins and perform 20 afﬁnity puriﬁcations.

Kit contents: UltraLink Iodoacetyl Spin Columns, 0.1mL

Coupling Buffer

L-Cysteine-HCl

Wash Solution

PBS Pack (makes 500mL)

IgG Elution Buffer

Collection Tubes

Kit

10 columns

100mL

100mg

25mL

1 pack

50mL

200 tubes

$357

For more information, or to download product instructions, visit www.thermoscientiﬁc.com/pierce

Thermo Scientiﬁc Disulﬁde Reducing Agents

Reduce disulﬁde bonds to produce sulfhydryl groups for

immobilization on SulfoLink or UltraLink Resins.

Free sulfhydryls are required for immobilization onto sulfhydryl-

reactive afﬁnity supports. Cysteines in proteins and peptides

usually exist as cystines (disulﬁde bridges) and must be reduced

to expose sulfhydryls for coupling. Reduction can be accomplished

with free or immobilized reducing agents. Free reducing agents

are efﬁcient in reducing all disulﬁdes in proteins, including those

buried in the tertiary structure, but they must be removed from the

reduced sample with a desalting column before coupling to the

support. Immobilized reducing agents enable reduction of disulﬁdes

and simple removal of the reduced sample from the reducing

agent. This is especially helpful when reducing peptides whose

small size prevents them from being effectively desalted.

–

TCEP•HCl

MW 286.65

–

2-Mercaptoethylamine•HCI

MW 113.61

DTT

MW 154.25

Ordering Information

Product #

Description

Pkg. Size

U.S.

Price

20408 2-Mercaptoethylamine•HCl

6 x 6mg

$135

20290 DTT, Cleland’s Reagent

(Dithiothreitol)

$105

20291 Dithiothreitol (DTT) in No-Weigh™ Format

7.7mg DTT/tube. Makes 100μL of 0.5 M DTT.

48 tubes

$125

20490 TCEP•HCl

(Tris[2-carboxyethyl]phosphine hydrochloride)

$ 50

20491 TCEP•HCl

10g

$299

77720 Bond-Breaker TCEP Solution, Neutral pH

5mL

$108

77712 Immobilized TCEP Disulﬁde Reducing Gel

5mL

$118

Thermo Scientiﬁc Ellman’s Reagent and Sulfhydryl Addition Kit

Measure and add free sulfhydryls to ensure success of

cysteine-targeted immobilization.

Ellman’s Reagent, also called DTNB, is a versatile water-soluble

compound for quantifying free sulfhydryl groups in solution. A

solution of this compound produces a measurable yellow-colored

product when it reacts with sulfhydryls (–SH groups). By testing

an unknown sample, such as a peptide having a terminal cysteine

residue, compared to a standard curve made with known amounts

of free, reduced cysteine (Product # 44889), availability of reduced

sulfhydryls in the sample can be determined.

The Sulfhydryl Addition Kit provides the essential reagents and

procedure for creating new sulfhydryl groups on a protein or

other molecule that contains available primary amines (–NH

The kit uses SATA reagent, which forms covalent bonds to primary

amines. The result is addition of a stable (capped) sulfhydryl

group, which can later be exposed by gentle treatment with

hydroxylamine, making the molecule ready for conjugation to

SulfoLink Coupling Resin, UltraLink Iodoacetyl Resin or other

sulfhydryl-reactive immobilization method.

Ordering Information

Product #

Description

Pkg. Size

U.S.

Price

23460 Sulfhydryl Addition Kit

Adds free sulfhydryl groups to proteins.

Includes: SATA

Hydroxylamine•HCl

10X Conjugation Buffer Stock

Phosphate Buffered Saline Pack

Dimethylformamide (DMF)

Dextran Desalting Column

Column Extender

Ellman’s Reagent (DTNB)

Cysteine•HCl H

Kit

2mg

5mg

20mL

1 pack

1mL

1 x 5mL

2mg

20mg

$223

22582 Ellman’s Reagent

(5,5’-Dithio-bis-[2-nitrobenzoic acid])

$ 74

44889 Cysteine•HCl

$ 45

Covalent Coupling of Afﬁnity Ligands

to Chromatography Supports

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch ofﬁce or distributor.

Products for Immobilizing Ligands

through Carbonyl Groups

Thermo Scientiﬁc CarboLink Immobilization Kit and

Coupling Resins

Immobilize polyclonal antibodies and other glycoproteins through

carbohydrate groups.

CarboLink Coupling Resin and Kits provide for covalent immobiliza-

tion of glycoproteins and other carbohydrate-containing molecules

to beaded agarose (or polyacrylamide UltraLink Support) for use

in afﬁnity puriﬁcation procedures. Carbohydrate moieties in glyco-

proteins contain common sugars whose cis-diol groups are easily

oxidized with sodium meta-periodate (included in the CarboLink

Kit) to yield aldehydes. When incubated with the CarboLink Resin,

these aldehyde groups react spontaneously with the hydrazide

group of the activated resin to form stable, covalent bonds. The

immobilization strategy is especially useful for glycoproteins,

such as polyclonal antibodies, because it allows attachment of the

molecule at domains that will not interfere with binding sites that

are critical for the intended afﬁnity puriﬁcation. Once a molecule

is coupled, the prepared afﬁnity resin can be used multiple times in

typical protein afﬁnity puriﬁcation procedures.

Highlights:

• CarboLink Coupling Resin – hydrazide-activated crosslinked

6% beaded agarose (or hydrazide-activated UltraLink Support,

a beaded, polyacrylamide resin)

• Efﬁcient immobilization – couple 1–5mg of oxidized polyclonal

antibody or other glycoprotein per milliliter of resin (CarboLink

Resin contains greater than 14μmol hydrazide groups per milliliter)

• Stable linkage – resonance structure of the hydrazone bonds

are sufﬁciently stable to allow multiple rounds of afﬁnity

puriﬁcation with one batch of prepared resin; no stabilizing

reductant required

• Flexible and gentle coupling conditions – immobilization reaction

completed in simple buffers (PBS or other non-amine buffer with

or without organic solvent) at near-neutral pH

• Ideal for polyclonal antibodies – immobilizes IgG through

carbohydrates in the Fc region, so both antigen binding sites are

free to interact with the antigen in the mobile phase

• Effective for any molecule with oxidizable sugars – ﬁrst step is

oxidation of the sugar groups, which allows the cis-diols of the

IgG to be transformed into reactive aldehyde moieties; these

aldehydes then combine with hydrazide groups on the matrix to

form stable, leak-resistant linkages

• Convenient kits and product sizes – choose one- or ﬁve-column

kit containing complete sets of buffers, oxidizing reagent and

versatile spin/drip columns containing the beaded agarose resin;

or choose the polyacrylamide-based UltraLink Support. Bulk

quantities are available for manufacturing applications

Agarose

Bead

Agarose

Bead

Thermo Scientific

CarboLink Resin

23-atom

Spacer

Hydrazide

Group

Antibody with Carbohydrate

Groups Oxidized to Form

Aldehyde Groups

Immobilized Glycoprotein

Oxidized Glycoprotein

Hydrazone

Bond

Thermo Scientiﬁc CarboLink Support immobilization chemistry.

Target: –CHO

Support: 6% agarose

Binding capacity: 5mg protein/mL resin

Time: 8 hours

References

1. Kumar, P.G., et al. (2001). J. Biol. Chem. 276, 41357–41364.

2. Strakova, Z., et al. (1997). Mol. Pharmacol. 51, 217–224.

3. Brown, M.A., et al. (2000). J. Biol. Chem. 275, 19795–19802.

4. Butko, P., et al. (1999). J. Immunol. 163, 2761–2768.

5. Sequra, M., et al. (1999). Infect. Immun. 67(9), 4646

Ordering Information

Product #

Description

Pkg. Size

U.S.

Price

20355 CarboLink Immobilization Trial Kit

Kit contains: CarboLink Column

CarboLink Coupling Buffer

CarboLink Wash Solution

Sodium meta periodate

Zeba Desalting Column

Kit

1 x 2mL

60mL

15mL

1 x 5mg

1 x 5mL

$155

44910 CarboLink Immobilization Kit

Kit contains: CarboLink Columns

CarboLink Coupling Buffer

CarboLink Wash Solution

Sodium meta periodate

Zeba Desalting Columns

Kit

5 x 2mL

250mL

100mL

5 x 5mg

5 x 5mL

$454

20391 CarboLink Coupling Resin

10mL

$113

53149 UltraLink Hydrazide Resin

10mL

$186

20504 Sodium meta-Periodate

25g

$ 29

For more information, or to download product instructions, visit www.thermoscientiﬁc.com/pierce

Products for Immobilizing Ligands

through Carboxyl Groups

Thermo Scientiﬁc CarboxyLink Immobilization Kits

and Coupling Resins

Immobilize peptides via carboxyl groups to create an afﬁnity column.

CarboxyLink Coupling Resin and Kits provide for covalent immobil-

ization of peptides or other carboxyl-containing (–COOH) molecules

to a porous, beaded resin for use in afﬁnity puriﬁcation procedures.

CarboxyLink Resin is crosslinked beaded agarose (or polyacryl-

amide UltraLink Support) that has been activated with diamino-

dipropylamine (DADPA) to contain long spacer arms, each with

a primary amine at the end. When incubated with the resin and

the carbodiimide crosslinker EDC (included in the CarboxyLink

Immobilization Kit), carboxyl-containing molecules become per-

manently attached to the support by stable amide bonds. Once

a molecule is coupled, the prepared afﬁnity column can be used

multiple times in typical protein afﬁnity puriﬁcation procedures.

CarboxyLink Coupling Resins can also be used to immobilize other

kinds of molecules using alternative amine-reactive crosslinking

chemistries.

Highlights:

• CarboxyLink Coupling Resin – DADPA-activated crosslinked

4% beaded agarose (or DADPA-activated UltraLink Support,

a beaded polyacrylamide resin)

• Efﬁcient immobilization – couple 1–2mg of peptide per milliliter

of resin (CarboxyLink Agarose Resin activated with greater than

16μmol amine milliliter of resin; DADPA on UltraLink Support

activated with greater than 40μmol amine milliliter of resin)

• Stable linkage – immoblization results in covalent attachment of

carboxyl groups by amide bonds, allowing for multiple rounds of

afﬁnity puriﬁcation with one batch of prepared resin

• Flexible and gentle coupling conditions – immobilization reaction

completed in simple MES or other non-amine and non-carboxyl,

near-neutral buffer, with or without organic solvent.

• Ideal for unmodiﬁed peptides – immobilizes peptides with

high capacity and various orientations without steric

hindrance, allowing for effective use in afﬁnity puriﬁcation of

speciﬁc antibodies

• Convenient kits and product sizes – choose ﬁve-column kits with

complete sets of buffers, crosslinker and versatile spin/drip

columns containing either type of resin (agarose or

polyacrylamide) or choose stand-alone resin for other uses

Immobilized DADPA

(Thermo Scientific CarboxyLink Resin)

EDC Crosslinker

Carboxyl Ligand

(e.g., peptide C-terminus)

Covalently Immobilized Ligand

(attached by amide bond and long spacer arm)

Agarose

Bead

Peptide

Agarose

Bead

Thermo Scientiﬁc CarboxyLink Support immobilization chemistry.

Target: –COOH

Support: 4% agarose or UltraLink

Resin

Binding capacity: 5mg protein/mL resin

Time: 4 hours

Reference

Yoo, B.C., et al. (2002). J. Biol. Chem. 277, 15325–15332.

Ordering Information

Product #

Description

Pkg. Size

U.S.

Price

44899 CarboxyLink Immobilization Kit

Kit contains: CarboxyLink Columns (DADPA agarose)

EDC Crosslinker

Coupling Buffer (MES-buffered Saline)

Wash Solution (1 M NaCl)

Column Accessories

Kit

5 x 2mL

5 x 60mg

500mL

120mL

$393

20266 CarboxyLink Coupling Resin

Support: Crosslinked 4% beaded agarose

Loading: 16–20μmol available amino groups/mL of resin

25mL

$145

53154 CarboxyLink Immobilization Kit

with UltraLink Support

Kit contains: DADPA UltraLink Columns

EDC Crosslinker

Coupling Buffer (MES-buffered Saline)

Wash Solution (1 M NaCl)

Column Accessories

Kit

5 x 2mL

5 x 60mg

500mL

120mL

$432

22980 EDC

1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide

hydrochloride

$ 83

28390 BupH MES Buffered Saline Packs

10 pack

$112

Covalent Coupling of Afﬁnity Ligands

to Chromatography Supports

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch ofﬁce or distributor.

Antibody immobilization: choosing the best Thermo Scientiﬁc Support.

AminoLink Plus

Coupling Resin

UltraLink

Biosupport

CarboLink

Coupling Resins

SulfoLink

Coupling Resins

Crosslink IP Kits

See page 41

Monoclonal Antibodies

Advantages:

• Good choice when

only small amounts of

antibody are available

• Couple over a broad

pH range

• Good coupling efﬁciency

Disadvantages:

• Reduction of Schiff’s base

with sodium cyanoborohy-

dride may adversely affect

monoclonals

• Some antibodies may

be coupled through

antigen-binding site

Advantages:

• Good choice if antibody

can withstand 1.0M

sodium citrate or sulfate

• High capacity

• Fast, efﬁcient coupling

• Good, for large-scale or

fast-ﬂow applications

Disadvantages:

• Some antibodies may be

coupled through antigen-

binding site

• Some antibodies may pre-

cipitate in high-salt buffer

Advantages:

• Correctly orients antibody

• Antibody must be able

to withstand oxidation

conditions

• Good for antibodies with

low avidity for antigen

Disadvantages:

• Not all monoclonals have

carbohydrate accessible

for coupling

• Conditions necessary for

coupling may adversely

affect some monoclonals

Advantages:

• Good choice for

antibodies that have

extremely high avidity

for their antigen

• Allows for gentle elution

conditions

Disadvantages:

• Must ﬁrst reduce antibody

prior to coupling

• Not good for antibodies

with low afﬁnity for

their antigens

Advantages:

• Allows for correct

orientation of antibodies

• Gentle coupling conditions

• Protein A/G will bind

most antibodies

Disadvantages:

• If purifying antigen from

serum, antibodies may

bind to Protein A/G and

co-purify with antigen

• Crosslinking results in

some loss of antibody

activity

Polyclonal Antibodies

Advantages:

• Excellent coupling

efﬁciency

• Good antigen recovery

Disadvantages:

• Some antibodies may

be coupled through

antigen-binding site

Advantages:

• Good choice for

large-scale or fast-ﬂow

applications

• High capacity

• Fast, efﬁcient coupling

Disadvantages:

• Some antibodies may

be coupled through

antigen-binding site

• Some antibodies may

precipitate in high-salt

buffer

Advantages:

• Correctly orients antibody

• Antibody must be able

to withstand oxidation

conditions

• Good for antibodies with

low avidity for antigen

Disadvantages:

• Conditions necessary for

coupling may adversely

affect some antibodies

Advantages:

• Good choice for anti-

bodies that have avidity

for their antigen

• Allows for gentle elution

conditions

Disadvantages:

• Must ﬁrst reduce antibody

prior to coupling

• Not good for antibodies

with low afﬁnity for their

antigens

Advantages:

• Allows for correct

orientation of antibodies

• Gentle couple conditions

• Protein A/G will bind

most antibodies

Disadvantages:

• If purifying antigen from

serum, antibodies may

bind to Protein A/G and

co-purify with antigen

• Crosslinking results in

some loss of antibody

activity

High-Activity Antibodies

Advantages:

• Immobilization of reduced

antibody allows for

gentler elution conditions

Low-Activity Antibodies

Advantages:

• Correctly orients antibody

Disadvantages:

• Conditions necessary for

coupling may adversely

affect some monoclonals

Advantages:

• Allows for correct

orientation of antibodies

• Gentle couple conditions

• Protein A/G will bind

most antibodies

Disadvantages:

• If purifying antigen from

serum, antibodies may

bind to Protein A/G and

co-purify with antigen

• Crosslinking results in

some loss of antibody

activity

For more information, or to download product instructions, visit www.thermoscientiﬁc.com/pierce

Immunoprecipitation

The topic of co-immunoprecipitation (co-IP) is best preceded by

a discussion of immunoprecipitation (IP) to help frame an under-

standing of the principles involved.

IP is one of the most widely used methods for antigen detection

and puriﬁcation. An important characteristic of IP reactions is

their potential to deliver not only the target protein, but also other

macromolecules that interact with the target.

The IP Principle

The principle of an IP is very simple (Figure 1). An antibody

(monoclonal or polyclonal) against a speciﬁc target antigen is

allowed to form an immune complex with that target in a sample,

such as a cell lysate. The immune complex is then captured on

a solid support to which either Protein A or Protein G has been

immobilized (Protein A or Protein G binds to the antibody, which

is bound to its antigen). The process of capturing this complex

from the solution is referred to as precipitation. Any proteins not

“precipitated” by the immobilized Protein A or Protein G support

are washed away. Finally, components of the bound immune

complex (both antigen and antibody) are eluted from the support

and analyzed by SDS-PAGE (gel electrophoresis), often followed

by Western blot detection to verify the identity of the antigen.

Coupled Antibody

Spin and wash

Cell Lysate or Protein Mixture

Incubation with

Antibody-coupled Resin

Antigen

Elute

Analyze

Figure 1. Summary of a traditional immunoprecipitation procedure.

Traditional immunoprecipitation involves the following steps:

1. Form the antigen-antibody complex (immune complex) by

incubating speciﬁc antibody with the antigen-containing sample

for 1 hour to several hours.

2. Capture the immune complex to Protein A or Protein G agarose

beads by incubation for 0.5-2 hours.

3. Remove any non-bound protein (non-immune complex sample

components) from the precipitated complex by washing beads

with additional sample buffer.

4. Boil beads in reducing SDS-PAGE sample loading buffer.

5. Recover eluted sample in loading buffer and analyze by

SDS-PAGE.

6. Perform Western blot analysis, probing with antigen-

speciﬁc antibody.

Co-IP vs. IP

Co-IP is a popular technique for protein interaction discovery.

Co-IP is conducted in essentially the same manner as IP (Figure 2).

However, in a co-IP the target antigen precipitated by the antibody

“co-precipitates” a binding partner/protein complex from a lysate;

i.e., the interacting protein is bound to the target antigen, which

becomes bound by the antibody that becomes captured on the

Protein A or Protein G resin. The assumption usually made when

associated proteins are co-precipitated is that these proteins are

related to the function of the target antigen at the cellular level. This

is only an assumption, however, that is subject to further veriﬁcation.

Coupled Antibody

Spin and wash

Cell Lysate or Protein Mixture

Incubation with

Antibody-coupled Resin

Antigen

Protein Interacting

with Antigen

Elute

Analyze

Figure 2. Summary of a traditional co-immunoprecipitation procedure.

IP/Co-IP

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch ofﬁce or distributor.

Traditional Methods vs.

Thermo Scientiﬁc Pierce Innovations for Co-IP

Problems with Traditional Co-IP Methods

The traditional co-IP protocol has certain deﬁciencies relating to

the fundamental format of the assay, the antibody and associated

chemistry. One of the most commonly encountered problems

with the traditional IP and co-IP approach is interference from

antibody bands in gel analysis. In those cases in which several

proteins may be co-precipitated with the target, presence of the

co-eluted antibody heavy and light chains (25 and 50 kDa bands

in reducing SDS-polyacrylamide gel) in the preparation can

obscure the results. The ideal situation would be to conduct

the co-IP without contamination of the eluted antigen with

antibody. With this potential interference eliminated, only the

co-precipitated proteins will be present and detected on a gel.

This and other shortcomings of the traditional protocol and our

solutions are summarized in Table 1.

Table 1. Comparison of traditional Co-IP and Thermo Scientiﬁc Pierce

Co-IP Products.

Traditional Co-IP Problems Thermo Scientiﬁc Pierce Product Solutions

Batch processing of the precipitated

complex in a single tube: results in

inefﬁcient washing of non-bound proteins

from the support and in resin loss due to

decanting wash buffer from tube via a

pipette, which lowers protein yields

Spin cup or spin tube processing:

dedicated IP and co-IP kits that

contain spin-cup or spin tube devices

that increase washing efﬁciency offer

more effective elution of antigen and

associated protein and eliminate resin

loss, yielding signiﬁcantly higher protein

recoveries and more consistent results

Antibody fragment interference:

co-elution of antibody fragments with

antigen often results in bands interfering

with detection of any co-precipitated

proteins on SDS-PAGE

Antibody immobilization: chemistries

designed to immobilize the antibody to

the support, thereby allowing elution

of only the target and any associated

proteins in a co-IP complex

Eliminate detection of antibody used

for immunoprecipitation: contaminating

antibody fragments become denatured

during SDS-PAGE. In contrast, the primary

antibody used during Western blotting is

still native. Use CleanBlot HRP Detection

Reagent (Product # 21230) to only detect

native antibody and not contaminants.

Antibody sacriﬁced: as a consequence

of harsh elution conditions, the target

antibody is destroyed; antibody loss by

way of the protocol can be costly

Antibody re-used: immobilization

chemistry and mild elution conditions

for the target and associated proteins

allow the immobilized antibody to be

re-equilibrated and recycled several times

in the co-IP protocol

Approaches to Co-IP Free of Antibody Interference

Three approaches have been incorporated into several products

targeted to IP and co-IP applications.

Activated Support for Antibody Immobilization — Direct Strategy

In this approach, an antibody is immobilized directly through its

surface amine groups (contributed primarily by the side chain

epsilon-amino group of lysine) to a high-capacity aldehyde-

activated beaded agarose support (Thermo Scientiﬁc AminoLink

Plus Coupling Resin). The support forms a Schiff’s base with these

available amines that is reduced to form stable secondary amine

bonds during the immobilization process. The wide range of coupling

conditions that can be used with this support make it ideal for

maintaining biological binding activity critical to the successful

execution of a co-IP experiment. When using the Direct Strategy,

the antibody source should be free of carrier protein, which can also

be immobilized. Products such as Thermo Scientiﬁc Melon Gel IgG

Puriﬁcation Kits (Product # 45206) can easily clean-up an antibody

to remove carrier proteins. The direct immobilization of antibodies is

not species dependent, which allows for the use on non-traditional

antibody sources that don’t bind globulin binding proteins (Protein A

or Protein G). Thermo Scientiﬁc Pierce Co-Immunoprecipitation Kits

(Product # 26149) and Thermo Scientiﬁc Pierce Direct IP Kit (Product

# 26148) use this direct immobilization approach. For co-IP applica-

tions, the ﬂexibility, simplicity and d urability of the direct method

as an antibody-coupling strategy makes it the method of choice for

delivering results free from antibody interference.

Antibody Orientation and Immobilization — Indirect Strategy

This strategy takes advantage of the binding characteristics of the

traditional Protein A or Protein G agarose combined with chemical

crosslinking to covalently link the antibody to the support. Protein

A and Protein G bind IgG class antibodies through the Fc region

that is characterized primarily by dimerized heavy chain modiﬁed

by carbohydrate. Fc region binding naturally orients the antigen-

binding domains of the antibody (Fab) away from the support,

making them available for binding to their respective target antigen.

The Indirect Strategy is compatible with antibody samples that

contain carrier proteins, provided the carrier proteins are washed

away prior to crosslinking. To ensure that the antibody remains on

the support during the requisite antigen binding, wash and elution

steps of the protocol, this bound and oriented antibody is chemi-

cally crosslinked to the Protein A or Protein G with the bifunctional

reagent disuccinimidyl suberate (DSS). Thermo Scientiﬁc Pierce

Crosslink IP Kit (Product # 26147) incorporates this strategy.

For more information, or to download product instructions, visit www.www.thermoscientiﬁc.com/pierce

Use of the Streptavidin:Biotin Interaction

This direct coupling approach incorporates the binding association

between streptavidin and biotin. Streptavidin immobilized to beaded

agarose resin or coated in microplate wells provides an alternative

IP or co-IP strategy for obtaining results free from antibody inter-

ference. Biotinylated antibody is bound very strongly to each matrix

and is not eluted when mild conditions are used to release the

target antigen. The IP/co-IP is conducted by incubating the sample

with the biotinylated antibody-loaded matrix. Elution of the target

antigen and any interacting proteins is performed free of antibody

contamination. Thermo Scientiﬁc Pierce Streptavidin Agarose Resin

(e.g., Product # 20347) and kit products that use this support, as well

as the Thermo Scientiﬁc Pierce Streptavidin Coated Plate IP Kit

(Product # 45360), provide high-capacity biotin-binding matrices

suitable for IP and co-IP applications.

Optimization Parameters in IP and Co-IP

Classical Immune Complex Formation vs. Pre-Binding of Antibody

A change in protocol from the classical immune complex

precipitation is necessary when using immobilized antibody

in the co-IP method. In the traditional co-IP protocol, the

immune complex (antigen:antibody) is formed in solution before

“precipitating” it with the immobilized Protein A or Protein G

matrix. When using immobilized antibody, the immune complex

is formed directly on the antibody-coupled matrix by incubation

of the antigen-containing sample with the matrix. Formation of the

immune complex (the target antigen and any target-associated

protein) and its precipitation occurs in one step.

In the immobilized format, the antibody is allowed to incubate

with the lysate. The matrix is washed using a spin cup format

and the bound protein eluted for analysis. The target antigen and

co-IP complex is recovered free of antibody or antibody fragment

contamination, and the antibody is retained in an active form on

the support to be used in another co-IP cycle.

Our research indicates that pre-binding or -coupling the antibody

to the support matrix consistently results in the capture of more

target antigen, even in coated plate IP procedures that do not

require it. This approach is recommended for the Thermo Scientiﬁc

Pierce Coated Plate IP Kits that use 96-well microplates coated

with streptavidin or Protein A/G (Products # 45360, 45350,

respectively). Antibody is bound to the plate wells prior to the

prescribed incubation with a lysate sample. Unbound protein is

easily washed from the wells prior to the elution of the target

and any co-precipitated proteins.

Evaluating a Co-IP-Captured Interaction

In their review of protein interactions, Phizicky and Fields

(see References listed below) present a discussion of the issues to

consider in validating a suspected interaction obtained by a co-IP

experiment. Ultimately, the following question must be answered:

Does the interaction detected by co-IP occur in vivo, and what

signiﬁcance does it have at the cellular level? A summary of the

Phizicky and Fields approach to veriﬁcation of co-IP data follows.

Conﬁrm that the co-precipitated protein is obtained only

by antibody against the target

Use monoclonal antibodies in the co-IP protocol. When only a

polyclonal antibody is available, pre-treatment of the antibody with

sample devoid of the primary target (bait protein) may be required

to assure that the polyclonal antibody does not contain clones or

contaminants that bind prey protein(s) directly. Pre-adsorption to

extracts devoid of target or pre-puriﬁcation of polyclonal IP

antibodies against an afﬁnity column containing pure target

antigen safeguards against a false-positive co-IP.

Conclude that antibody against the target antigen does not

itself recognize the co-precipitated protein(s)

Use independently derived antibodies that have demonstrated

speciﬁcities against different epitopes on the target protein.

Their use serves as veriﬁcation that the target (bait)-directed

antibodies have no afﬁnity for the target-associated prey proteins

recovered during the co-IP. Alternatively, an antibody against the

co-precipitated protein can be used to co-IP the same complex.

Determine if the interaction is direct or indirect

Is the interaction mediated through a third-party protein that

contacts both target and co-precipitated protein? Immunological

and other more sophisticated methods such as mass spectrometry

may be necessary to answer this question.

Determine that the interaction takes place in the cell and not

as a consequence of cell lysis

Suggested approaches here involve co-localization studies and

site-speciﬁc mutagenesis giving rise to mutants that perturb the

binding process.

References

Adams, P.D., et al. (2002). Identiﬁcation of associated proteins by co-immunoprecipitation,

In Protein-Protein Interactions – A Molecular Cloning Manual. Golemis, E., Ed., Cold Spring

Harbor Laboratory Press, pp 59-74.

Liebler, D.C. (2002). Identifying protein-protein interactions and protein complexes.

In Introduction to Proteomics, Tools for the New Biology, Humana Press, pp.151-165.

(Product # 20061)

Phizicky, E.M. and Fields, S. (1995). Protein-protein interactions: methods for detection

and analysis. Microbiological Reviews (Mar.), pp. 94-123.

IP/Co-IP

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch ofﬁce or distributor.

IP and Co-IP Kits

The Thermo Scientiﬁc Pierce Classic and Crosslink IP Kits are

ideal when using antibodies that bind to Protein A or Protein G.

The Direct and Crosslink IP Kits are highly effective for eliminating

co-elution of IgG heavy and light chain with the antigen, which

interferes with downstream applications such as mass

spectrometry analysis or protein sequencing.

Traditional co-IP methods result in detection of the antibody with

the target proteins. Because the antibody heavy and light chains

may co-migrate with one of the relevant bands, important results

can be masked. The Pierce Co-IP Kit circumvents issues with

co-migration of antibody chains with target proteins by retain-

ing the antibody on the resin. The new kit is optimized for using

smaller amounts of sample and offers a single lysis/wash buffer

eliminating the need for a separate lysis reagent.

All four IP Kits Highlights:

• Require minimal antibody (2-10μg)

• Are highly effective and efﬁcient in capturing antigens

• Use optimized protocols and buffers for efﬁcient IPs and

antigen elution

• Use common lysis/binding/wash buffer

• Include spin columns and collection tubes that shorten the

protocol by minimizing handling and mixing

• Use a new elution buffer that provides milder and less denaturing

recovery of antibody:antigen complexes

• Are compatible with specialized downstream applications; e.g.,

mass spectrometry, enzyme assays and antibody production

• Are able to scale up as needed using our ﬂexible protocol

Comparison of immunoprecipitation methods. The following table compares the key features of traditional “do-it-yourself” immunoprecipitation techniques

to the Thermo Scientiﬁc Pierce IP Kits. Consideration of these features can help to determine which method is most appropriate for the available reagents and

downstream application.

Feature

Traditional

IP method

Pierce

Classic IP Kit

Pierce

Crosslink IP Kit

Pierce

Direct IP Kit

Pierce

Co-IP Kit

Uses high binding capacity resin Variable

Yes

(Protein A/G Plus)

Yes

(Protein A/G Plus)

Yes

(AminoLink Plus)

Yes

(AminoLink Plus)

Crosslinker mediated immobilization No No Yes

(DSS)

No No

Requires puriﬁed antibody in amine-free and protein-free

storage solution

No No No Yes Yes

Antibody is covalently attached to agarose resin No No Yes Yes Yes

Antibody is oriented Yes Yes Yes No No

Antibody elutes with antigen Yes Yes No No No

Antigen recovery method Boiling w/SDS

(Low pH)

Low pH elution

(Boiling w/SDS)

Low pH

elution

Low pH

elution

Low pH

elution

Relative antigen recovery

Variable Highest Medium High High

Immobilized antibody can be reused No No Possible

Possible

Co-puriﬁcation of interacting proteins Possible Possible Possible Optimal

Commercially available resins vary in binding capacity and performance for IP assays.

Antigen yield depends on the activity of the antibody, speciﬁc binding conditions and the immobilization method used.

It is possible to reuse the prepared antibody afﬁnity resin if the antibody remains functional following low pH elution.

For more information, or to download product instructions, visit www.www.thermoscientiﬁc.com/pierce

Comparison of three different Thermo Scientiﬁc Pierce IP Kits.

Immunoprecipitations were performed according to the product instructions

using 10μg of afﬁnity-puriﬁed goat anti-GFP antibody and the Pierce Direct,

Classic and Crosslink IP Kits. The cell lysate was prepared using IP Lysis/

Wash Buffer and pre-cleared using the Pierce Control Agarose Resin supplied

in the kits. The immune complex was formed by incubating the antibody, resin

and lysate overnight. The resin was washed with IP Lysis/Wash Buffer, 1X

Conditioning Buffer and eluted with Elution Buffer. For analysis, 4-20%

Tris-glycine gels were loaded with 20% of the eluted sample, 5% of the cell

lysate load (Lysate) and 10% of the antibody load (IgG) and stained with

Thermo Scientiﬁc Imperial Protein Stain (Product # 24615). For the resin con-

trols, the immunoprecipitation was performed without adding the antibody.

Co-immunoprecipitation of interacting proteins using the Thermo Scientiﬁc

Pierce Co-IP Kit. Epidermal Growth Factor (EGF) was co-immunoprecipitated

from 2,000μg of HeLa lysate with 40 and 20μg anti-Epidermal Growth Factor

Receptor (EGFR) mouse monoclonal IgG

+ IgG

(Thermo Scientiﬁc) using

the Pierce Co-IP Kit. Co-immunoprecipitations were performed according to

product instructions. Two identical Western blots were probed with anti-EGFR

sheep polyclonal IgG (Millipore) or anti-EGF rabbit polyclonal IgG (Santa Cruz).

The Pierce Co-IP Kit co-immunoprecipitated EGF with the EGF-R.

The Classic IP Kit:

Product # 26146

• High binding-capacity recombinant Protein

A/G Plus Agarose Resin results in higher

antigen yields

• Recombinant Protein A/G Plus Resin offers

compatibility with a wider range of mamma-

lian IgG species for IP reactions (e.g., mouse,

rabbit, human and goat IgG subclasses)

The Crosslink IP Kit:

Product # 26147

• Able to purify target protein without con-

tamination by the antibody in the eluate

• Improved crosslinking protocol optimized

for maximum antibody functionality

• Bound immunoglobulins oriented for optimal

antigen-binding sites are more accessible

• High-capacity Protein A/G Plus Resin allows for better puriﬁ-

cation and immobilization of antibodies

• Offers compatibility with a wider range of mammalian

IgG species

The Direct IP Kit:

Product # 26148

• Immobilize any antibodies independent

of isotype or species

• Improved antibody coupling protocol

• Purify target protein without antibody

contamination

• Activated resin for directly coupling antibodies to the

support resin

• Eliminate antibody contamination in the eluate

The Co-IP Kit:

Product # 26149

• Minimal antibody requirements for

co-IP reactions

• Shorter antibody coupling protocol

(<2 hours)

• Compatible with any antibody species

and subclass

• Requires a puriﬁed antibody in a solution free of amines and

stabilizing proteins

• Optimized protocols and buffers for efﬁcient co-IP and

antigen elution

• Allows for selective puriﬁcation of target protein

• Includes spin columns and collection tubes that shorten the

protocol by minimizing handling and mixing

• Compatible with specialized downstream applications,

e.g., mass spectrometry

• Allows for scale up

Bead

Protein A/G Plus

Antibodies

Alkylamine linkage

DSS Crosslinking

GFP (27 kDa)

IgG Heavy Chain

(55kDa)

Thermo Scientific

Pierce IP Kits

Direct

Classic

Lysate

IgG

Direct

Classic

Crosslink

IgG Light Chain

(25kDa)

Resin Controls

EGF-R (170kDa)

µg anti-EGFR

EGF (6kDa)

40 20

IP/Co-IP

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch ofﬁce or distributor.

Thermo Scientiﬁc Pierce Classic IP Kit

A convenient kit for a new spin on traditional immunoprecipitation.

The Pierce Classic IP Kit provides all the necessary reagents,

spin cups and collection tubes to perform successful immunopre-

cipitation (IP) experiments with ease. The kit uses high-capacity

Protein A/G agarose afﬁnity resin for efﬁcient binding of most

species and subclasses of IP antibodies. The included IgG elu-

tion buffer provides milder and less denaturing recovery of

antibody:antigen complexes than the traditional method of boiling

in reducing sample buffer for SDS-PAGE, facilitating a greater

variety of methods for subsequent analysis. The microcentrifuge

spin column format helps to ensure effective washing and sep-

aration of samples from the beaded agarose afﬁnity resin.

Like traditional IP methods, the Pierce Classic IP Kit procedure

involves formation of antibody:antigen complexes in a sample

solution and then capture of that complex to an IgG-binding

protein that is covalently bound to beaded agarose resin (Protein

A/G Agarose). After washing to remove nonbound (presumably

undesired) components of the sample, the antigen and antibody

are recovered from the beaded resin with elution buffer supplied

in the kit. The entire procedure is performed in a microcentrifuge

spin column, allowing solutions to be fully separated from the

agarose resin upon brief centrifugation.

Ordering Information

Product #

Description

Pkg. Size

U.S.

Price

26146 Pierce Classic IP Kit

Sufﬁcient reagents to perform 50 reactions.

Includes: Pierce Protein A/G Plus Agarose

IP Lysis/Wash Buffer

100X Conditioning Buffer

20X Tris-Buffered Saline

Elution Buffer

5X Lane Marker Sample

Buffer, Non-reducing

Pierce Spin Columns – Screw Cap

Microcentrifuge Collection Tubes

Microcentrifuge Sample Tubes

Pierce Control Agarose Resin

Kit

0.55mL

2 x 50mL

5mL

25mL

50mL

5mL

50 each

2mL, 100 each

1.5mL, 50 each

2mL

$259

Thermo Scientiﬁc Pierce Crosslink IP Kit

Purify target protein complexes without antibody interference!

The Crosslink IP Kit extends the functionality of traditional immuno-

precipitation (IP) methods by adding crosslinking technology and

microcentrifuge spin column sample handling to the procedure.

The primary beneﬁts resulting from these features are the ability

to purify target protein without contamination by the antibody and

the ability to more effectively wash and separate samples from the

beaded agarose resin.

The Pierce Crosslink IP Kit method involves capturing the IP anti-

body to Protein A/G Agarose resin and covalently immobilizing it

to the support by crosslinking with disuccinmidyl suberate (DSS).

The antibody resin is then incubated with the sample that contains

the protein antigen of interest, allowing the antibody:antigen com-

plex to form. After washing to remove nonbound (presumably

undesired) components of the sample, the antigen is recovered by

dissociation from the antibody with elution buffer supplied in the kit.

The entire procedure is performed in a microcentrifuge spin cup,

allowing solutions to be fully separated from the agarose resin

upon brief centrifugation. Only antigen is eluted by the procedure,

enabling it to be identiﬁed and further analyzed without interfer-

ence from antibody fragments. Furthermore, the antibody resin

often can be reused for additional rounds of immunoprecipitation.

Ordering Information

Product #

Description

Pkg. Size

U.S.

Price

26147 Pierce Crosslink IP Kit

Sufﬁcient reagents to perform 50 reactions.

Includes: Pierce Protein A/G Plus Agarose

20X Coupling Buffer

DSS Crosslinker, No-Weigh

™

Format

IP Lysis/Wash Buffer

100X Conditioning Buffer

20X Tris-Buffered Saline

Elution Buffer

Lane Marker Sample Buffer,

Non-reducing, (5X)

Pierce Spin Columns - Screw Cap

Microcentrifuge Collection Tubes

Microcentrifuge Sample Tubes

Pierce Control Agarose Resin

Kit

0.55mL

25mL

8 x 2mg

2 x 50mL

5mL

25mL

50mL

5mL

50 each

2mL, 100 each

1.5mL, 50 each

2mL

$305

For more information, or to download product instructions, visit www.www.thermoscientiﬁc.com/pierce

Thermo Scientiﬁc Pierce Direct IP Kit

Immunoprecipitate using any antibody species or subclass!

Eliminate antibody band contamination of IP products.

The Pierce Direct IP Kit represents a signiﬁcant advancement in

immunoprecipitation (IP) technology by replacing the use of

immobilized Protein A or Protein G with a method for direct covalent

attachment of antibodies to the beaded agarose resin.

The primary beneﬁts resulting from this method are the opportunity

to use any species or subclass of puriﬁed antibody (not just types

that bind to Protein A or G) and the ability to purify target protein

without contamination by the antibody. The method also makes

it possible to immunoprecipitate antigens from serum samples

without co-purifying non-target immunoglobulins. Finally, the kit

uses microcentrifuge spin cups to effectively wash and separate

samples from the beaded agarose resin.

Ordering Information

Product #

Description

Pkg. Size

U.S.

Price

26148 Pierce Direct IP Kit

Sufﬁcient reagents to perform 50 reactions.

Includes: AminoLink Plus Coupling Resin

20X Coupling Buffer

Quenching Buffer

Wash Solution

5M Sodium Cyanoborohydride Solution

IP Lysis/Wash Buffer

100X Conditioning Buffer

20X Tris-Buffered Saline

Elution Buffer

5X Lane Marker Sample Buffer

Pierce Spin Columns – Screw Cap

Microcentrifuge Collection Tubes

Microcentrifuge Sample Tubes

Pierce Control Agarose Resin

Kit

2mL

25mL

50mL

0.5mL

2 x 50mL

5mL

25mL

50mL

5mL

50 each

2mL, 100 each

1.5mL, 50 each

2mL

$259

Thermo Scientiﬁc Pierce Co-Immunoprecipitation Kit

Perform co-immunoprecipitation experiments without

antibody interference.

The Pierce Co-Immunoprecipitation (Co-IP) Kit enables isolation

of native protein complexes from a lysate or other complex

mixture by directly immobilizing puriﬁed antibodies onto an

agarose support.

Co-IP is a common approach to study protein:protein interac-

tions that uses an antibody to immunoprecipitate the antigen (bait

protein) and co-immunoprecipitate any interacting proteins (prey

proteins). Traditional co-IP methods that use Protein A or G result

in co-elution of the antibody heavy and light chains that may

co-migrate with relevant bands, masking important results. The

Pierce Co-IP Kit resolves this issue by covalently coupling anti-

bodies onto an amine-reactive resin. The kit includes optimized

buffers for protein binding and recovery, reagents to perform con-

trol experiments and efﬁcient spin columns and collection tubes,

which shorten the protocol and minimize handling and mixing.

Ordering Information

Product #

Description

Pkg. Size

U.S.

Price

26149 Pierce Co-Immunoprecipitation Kit*

Sufﬁcient reagents to perform 50 reactions.

Includes: AminoLink Plus Coupling Resin

20X Coupling Buffer

5M Sodium Cyanoborohydride Solution

Quenching Buffer

Wash Solution

IP Lysis/Wash Buffer

100X Conditioning Buffer

Elution Buffer

5X Lane Marker Sample

Buffer, Non-reducing

Pierce Control Agarose Resin

20X Modiﬁed Dulbecco’s PBS Buffer

Pierce Spin Columns – Screw Cap

Microcentrifuge Collection Tubes

Microcentrifuge Sample Tubes

Kit

2mL

25mL

0.5mL

50mL

60mL

2 x 50mL

5mL

50mL

5mL

2mL

25mL

50 each

100 each

50 each

$308

* The Thermo Scientiﬁc Pierce Co-IP Kit (Product # 23600) and the Pierce Mammalian Co-IP Kit (Product

# 23605) were discontinued on December 31, 2009. The next-generation Pierce Co-IP Kit (Product #

26149) will replace these old kits. The new co-IP kit is optimized for using smaller amounts of sample and

offers a common lysis/wash buffer eliminating the need for a separate lysis reagent (Thermo Scientiﬁc

M-PER Mammalian Protein Extraction Reagent, Product # 78503) as was offered with the Mammalian

Co-IP Kit.

IP/Co-IP

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch ofﬁce or distributor.

Thermo Scientiﬁc Pierce Coated Plate Immunoprecipitation Kits

Pre-coated 96-well plates are easier to use and faster than

traditional microcentrifuge tube methods.

Thermo Scientiﬁc Pierce Coated Plate IP Kits enable rapid

immunoprecipitation of multiple samples without the usual tedium

of pipetting, centrifuging and separating beaded afﬁnity resin

in individual microcentrifuge tubes. Immunoprecipitation is

accomplished using coated 96-well microplates rather than

beaded agarose resin. The plate format allows fast processing

of multiple samples. Select from Protein A/G-, Protein G- or

streptavidin-coated plates.

Highlights:

• Ready-to-use, high-quality coated plates provide high capacity

and consistency

• Plate format best suited for simultaneously processing multiple

samples and their control conditions

• Faster, easier and more thorough washing than with traditional

tube/resin IP methods

• Uses familiar and convenient ELISA tools (multichannel pipettors

and plate washing); no tedious separation of supernatant from

pelleted resin beads, and no tubes to open, close and centrifuge

• Coated plates are 96-well strip plates, convenient for experi-

ments requiring only a partial plate

• Easy-to-follow instructions, including detailed explanation of

appropriate controls

• Three kits available, suitable for most common antibody types

(mouse, rabbit, human and goat IgG subclasses) or any

biotinylated antibody or “bait” protein

3 4 521

Thermo Scientiﬁc Pierce Protein A/G Coated Plate IP Kit (Product # 45350)

immuno precipitation of CD71 (transferring receptor) from human serum using

and a goat anti-CD71 polyclonal antibody. Eluted products for the experimental

and control samples were mixed with nonreducing sample loading buffer,

separated by SDS-PAGE, transferred to nitrocellulose membrane and detected

by Western blotting with the IP antibody, Goat-anti-mouse-HRP conjugated

secondary antibody (Product # 31432) and Thermo Scientiﬁc SuperSignal West

Dura Chemiluminescent Substrate (Product # 34076).

Lane 1: Experiment (immunoprecipitation product)

Lane 2: Antibody-only control (no sample)

Lane 3: Human serum sample control (no antibody)

Lane 4: Plate control (no antibody or human sample)

Lane 5: Pure target protein (CD71) for size reference

Choosing between Protein A/G and Streptavidin Coated Plate Kits

Streptavidin is a protein that binds speciﬁcally and strongly to

biotin; therefore, the Streptavidin Coated Plate IP Kit

(Product # 45360) is appropriate for immunoprecipitation when

using a biotin-labeled (biotinylated) antibody. This kit can be

used to afﬁnity-purify a binding partner to any antibody species

or subclass or any other protein or molecule that is biotinylated.

Because the streptavidin-biotin afﬁnity interaction is so strong,

the elution step generally will dissociate only the antigen (binding

partner), not the biotinylated antibody or “bait” protein.

Protein A and Protein G are different proteins that bind to immuno-

globulins (primarily IgG). Typically, Protein A is preferred for use

with rabbit polyclonal antibodies, while Protein G is preferred for

use with mouse antibodies (especially monoclonals of the IgG

subclass). Protein A/G is a recombinant of Protein A and Protein G

that has the additive binding properties of both proteins.

Reference

Desai, S. and Hermanson, G. (1997). Previews 1(3), 2-7.

Ordering Information

Product #

Description

Pkg. Size

U.S.

Price

45350 Pierce Protein A/G Coated Plate IP Kit

Antibody binding capacity/well: 2.5µg. Sufﬁcient

capacity for downstream analysis of IP or co-IP

proteins in-gel or by Western blot.

Includes: Protein A/G Coated 12 x 8-well strip plates

Phosphate buffered saline

Surfact-Amps

X-100 (10% Triton X-100)

Elution buffer

Neutralization buffer

Uncoated 96-well strip plates (white),

(for sample collection and neutralization)

Plate sealers

Kit

2 plates

2 packs

6 x 10mL

50mL

7mL

2 ea.

18 sheets

$295

45360 Pierce Streptavidin Coated Plate IP Kit

Antibody binding capacity/well: 5µg. Sufﬁcient

capacity for downstream analysis of IP or co-IP

proteins in-gel or by Western blot.

Includes: High Binding Capacity Streptavidin

Coated Plates

Biotin blocking buffer

Phosphate buffered saline

Surfact-Amps X-100 (10% Triton X-100)

Elution buffer

Neutralization buffer

Uncoated 96-well strip plates (white),

(for sample collection and neutralization)

Plate sealers

Kit

2 plates

30mL

2 packs

6 x 10mL

50mL

7mL

2 ea.

18 sheets

$298

For more information, or to download product instructions, visit www.www.thermoscientiﬁc.com/pierce

Thermo Scientiﬁc Pierce HA- or c-Myc Tag IP/

Co-Immunoprecipitation Kits

Need to perform IP or co-IP reactions with your HA- or c-Myc-

tagged protein? Open box ... Read instructions ... Start performing

an IP or co-IP. High-speciﬁcity immobilized antibodies make it easy.

No tags are more popular for mammalian system protein expres-

sion than HA or c-Myc. Although these tags are extremely popular,

a kit that allows you to conveniently perform immunoprecipitation

(IP) or co-immunoprecipitation (co-IP) reactions using these tags

has not been available. The Pierce IP/Co-IP Kits include all neces-

sary reagents, buffers and hardware that allow efﬁcient puriﬁca-

tion of a tagged target protein (i.e., IP) or conﬁrmation of potential

interactions indicated by yeast two-hybrid results (i.e., co-IP).

These four kits, which are speciﬁcally for HA- and c-Myc-tagged

proteins, allow you to easily perform an IP or co-IP experiment

with minimal optimization. High-afﬁnity, high-speciﬁcity antibod-

ies immobilized onto an agarose matrix are at the heart of these

new kits. In addition, the kits contain a full complement of buffers,

eluents, a positive control and necessary hardware to efﬁciently

perform the intended application.

Highlights:

IP and co-IP for HA- or c-Myc-tagged proteins directly out of the box.

• Demonstrated utility in the IP and co-IP application beneﬁts the

novice and expert. Our kits include all essential components to

perform the assays. There’s no need to formulate or validate

raw materials.

Immobilized high-afﬁnity antibodies with excellent speciﬁcity

for the HA or c-Myc tag.

• The immobilized anti-HA and anti-c-Myc monoclonals precipitate

the appropriately tagged protein speciﬁcally and in high yield,

resulting in clean Western blot detection.

• Excellent results with as little as 2.5μg of anti-HA antibody and

1μg of anti-c-Myc antibody in the IP mode with the respective

positive control lysate.

• Limits possibility of nonspeciﬁc binding to other proteins in

the lysate.

• Eliminates contamination from antibody or antibody fragments

after elution of the precipitated protein or the co-IP complex.

This beneﬁt is especially important when interpreting protein

interaction results.

Simple, ﬂexible and easy-to-follow protocols.

• Complete kit format offers optimum convenience in both the

IP and co-IP modes.

• Eliminate Protein A or Protein G, reducing nonspeciﬁc binding

and shortening the IP procedure.

• System demonstrates excellent ﬂexibility with respect to the

amount of antibody or amount of lysate used, enabling isolation

of low-expression HA-/c-Myc-tagged targets.

• The use of Spin Columns accelerate the IP/co-IP process.

Spin Columns.

• Spin Columns are very convenient for small sample handling.

• Allow more efﬁcient washing.

• Eliminate resin losses

HA- or c-Myc Tag IP/Co-IP Kit Descriptions

Each kit listed at right consists of two components: an Application

Set and a Positive Control Lysate. The Application Set contains

the immobilized support appropriate for the kit and all of the

required buffers, eluents and hardware. The second component

is a bacterial lysate containing an overexpressed GST with

either HA or c-Myc as the C-terminal tag. The mammalian

version of each kit contains Thermo Scientiﬁc M-PER Protein

Extraction Reagent for use with mammalian cell-based IP or co-IP

applications. The Application Sets and Positive Control Lysates

can also be ordered separately.

1. Lyse cells expressing HA- or

c-Myc-tagged protein.

2. Combine cell lysate and

Immobilized Anti-HA or

Anti-c-Myc.

3. Incubate at 4

C for

1 hour to overnight with

end-over-end mixing.

4. Pulse centrifuge for

10 seconds.

5. Wash three times with

TBS-T. Pulse centrifuge for

10 seconds after each wash.

6. Elute HA- or c-Myc-tagged

protein using Elution Buffer

or Non-Reducing Sample Buffer.

Collection

Tube

Bottom Plug

Removed

Thermo Scientiﬁc Pierce HA- or c-Myc IP/Co-IP Kit Protocol summary.

GST-c-Myc

GST-HA

MW Marker

HA Lysate

Elution #1

Elution #2

c-Myc Lysate

Elution #1

Elution #2

Effectiveness of elution options in the IP of GST-HA and GST-c-Myc from

bacterial lysates. IP results achieved with the Thermo Scientiﬁc Pierce HA

and c-Myc IP/Co-IP Kits using the appropriate positive control lysate provided

and suggested elution options for GST-HA and GST-cMyc, respectively.

The elution components are supplied with each kit. Elution performed with

#1. Elution Buffer or #2. 2X nonreducing sample buffer.

IP/Co-IP

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch ofﬁce or distributor.

Ordering Information

Product #

Description

Pkg. Size

U.S.

Price

23610 HA-Tag IP/Co-IP Kit

Sufﬁcient material to conduct 25 IP/co-IP assays using

proteins expressed with an HA tag. Kit is supplied

complete with an HA-tagged positive control lysate.

Includes: Immobilized Anti-HA (agarose resin)

BupH Tris Buffered Saline Pack

Elution Buffer, pH 2.8

Lane Marker Non-Reducing Sample Buffer (5X)

Spin Columns Accessory Pack,

27 columns with pre-inserted frit

and top and bottom caps

Collection Tubes and Caps Accessory Pack,

100 graduated 2mL tubes and plug caps

M-PER Mammalian Protein Extraction

Reagent

HA-Tagged Positive Control

Kit

150μL

1 pack

50mL

5mL

25mL

500μL

$399

23615 Mammalian HA-Tag IP/Co-IP Kit

Sufﬁcient material to conduct 25 IP/co-IP assays using

proteins expressed with an HA tag. Kit is supplied

complete with a mammalian cell lysis buffer and

an HA-tagged positive control lysate.

Includes: Immobilized Anti-HA (agarose resin)

BupH

Tris Buffered Saline Pack

Elution Buffer, pH 2.8

Lane Marker Non-Reducing Sample Buffer (5X)

Spin Columns Accessory Pack,

27 columns with pre-inserted frit

and top and bottom caps

Collection Tubes and Caps Accessory Pack,

100 graduated 2mL tubes and plug caps

M-PER Mammalian Protein Extraction

Reagent

HA-Tagged Positive Control

Kit

150μL

1 pack

50mL

5mL

25mL

500μL

$423

23620 c-Myc-Tag IP/Co-IP Kit

Sufﬁcient material to conduct 25 IP/co-IP assays using

proteins expressed with a c-Myc tag. Kit is supplied

complete with a c-Myc-tagged positive control lysate.

Includes: Immobilized Anti-c-Myc (agarose resin)

BupH Tris Buffered Saline Pack

Elution Buffer, pH 2.8

Lane Marker Non-Reducing Sample Buffer (5X)

Spin Columns Accessory Pack,

27 columns with pre-inserted frit

and top and bottom caps

Collection Tubes and Caps Accessory Pack,

100 graduated 2mL tubes and plug caps

c-Myc-Tagged Positive Control

Kit

250μL

1 pack

50mL

5mL

500μL

$399

23625 Mammalian c-Myc Tag IP/Co-IP Kit

Sufﬁcient material to conduct 25 IP/co-IP assays using

proteins expressed with a c-Myc tag. Kit is supplied

complete with a mammalian cell lysis buffer and a

c-Myc-tagged positive control lysate.

Includes: Immobilized Anti-c-Myc (agarose resin)

BupH Tris Buffered Saline Pack

Elution Buffer, pH 2.8

Lane Marker Non-Reducing Sample Buffer (5X)

Spin Columns Accessory Pack,

27 columns with pre-inserted frit

and top and bottom caps

Collection Tubes and Caps Accessory Pack,

100 graduated 2mL tubes and plug caps

M-PER Mammalian Protein Extraction

Reagent

c-Myc-Tagged Positive Control

Kit

250μL

1 pack

50mL

5mL

25mL

500μL

$423

Protein Interactions Technical Handbook

Our 72-page Protein Interaction

Technical Handbook provides

protocols and technical and product

information to help maximize results

for Protein Interaction studies. The

handbook provides background,

helpful hints and troubleshooting

advice for immunoprecipitation and

co-immunoprecipitation assays, pull-

down assays, Far-Western blotting

and crosslinking. The handbook also

features an expanded section on

method to study protein:nucleic acid

interactions, including ChIP, EMSA and RNA EMSA. The hand-

book is an essential resource for any laboratory studying Protein

Interactions. (# 1601618)

For more information, or to download product instructions, visit www.thermoscientiﬁc.com/pierce

Protein Enrichment

Protein enrichment encompasses numerous techniques to

isolate subclasses of cellular proteins based on their unique

biochemical activity, post-translational modiﬁcation (PTM) or

spatial localization in a cell. Protein enrichment is essential for

studying low abundant proteins and for reducing the complexity

of samples for proteomic analysis. Enrichment of speciﬁc proteins

or protein complexes can most easily be accomplished using

immunoafﬁnity techniques such as immunoprecipitation and

co-immunoprecipitation. Although these antibody-based tech-

niques are widely used, elution of immunoprecipitated proteins

can sometimes result in low protein recovery or antibody contami-

nation in samples.

Global protein enrichment strategies involve the selective isola-

tion of distinct protein subclasses which share a common post-

translational modiﬁcation or cellular localization. Post-translational

modiﬁcations such as phosphorylation and glycosylation can be

enriched using afﬁnity ligands such as ion-metal afﬁnity chroma-

tography (IMAC) or immobilized lectins, respectively. In addition,

PTM-speciﬁc antibodies have been be used. Other techniques use

metabolic or enzymatic incorporation of modiﬁed amino acids or

PTMs to introduce unique protein chemistry which can be used for

enrichment. Finally, proteins can also be enriched using various

enzyme class speciﬁc compounds or cell-impermeable labeling

reagents which selectively label cell surface proteins.

Phosphoprotein Enrichment Kits

Process cell and tissue samples in less time and with greater purity.

Phosphorylation is one of the most frequently occurring post-

translational modiﬁcations in proteins. It is estimated that as many

as 30% of all cellular proteins are transiently phosphorylated on

serine, threonine and tyrosine residues.

Reversible protein phosphorylation regulates nearly all intra-

cellular biological events, including signal transduction, protein-

protein interactions, protein stability, protein localization, apoptosis

and cell-cycle control. Deregulation of protein phosphorylation is

a hallmark of numerous human diseases, including cancer and

metabolic and immune disorders.

Detecting changes in protein phosphorylation can be a difﬁcult

task because of the transient labile state of the phosphate

group. Furthermore, low phosphoprotein abundance and poorly

developed phospho-speciﬁc antibodies contribute to difﬁculties in

phosphoprotein detection. Recent advances in mass spectrometry

technology in combination with phosphoprotein enrichment using

immobilized metal afﬁnity chromatography (IMAC) have resulted in

greater resolution of the phosphoproteome.

The new Thermo Scientiﬁc Pierce Phosphoprotein Enrichment Kit

efﬁciently enriches phosphorylated proteins derived from mammalian

cells and tissues. The proprietary metal and buffer composition

produces superior yields with negligible nonspeciﬁc binding.

Highlights:

• Speciﬁc – low contamination from nonspeciﬁc proteins

• Fast – easy-to-use spin format enriches of phosphorylated

proteins in less than 2 hours

• Superior yield – high yield from complex biological samples, cell

culture lysate and mouse tissue extract

• Convenient format – complete kit includes pre-dispensed spin

columns, buffers, reagents and Thermo Scientiﬁc Pierce Protein

Concentrators

• Compatible – works with downstream applications, including

mass spectrometry, Western blotting and 2D-PAGE

Phospho-speciﬁc antibodies recognizing key regulatory proteins

involved in growth factor signaling were used to monitor binding

speciﬁcity of our Phosphoprotein Enrichment Kit (Figure 1).

Speciﬁcity of the kit is further demonstrated by the absence of

Cytochrome C (pI 9.6) and p15Ink4b (pI 5.5), two proteins not

predicted to be phosphorylated, in the elution fraction and their

emergence in the ﬂow-through and wash fractions (Figure 1).

Furthermore, dephosphorylation of HeLa cell extract in vitro

resulted in diminished binding of PTEN, MAPK and GSK3β to the

Pierce Phosphoprotein Enrichment Column as evidenced by their

absence in the elution fraction. Conversely, all three proteins

were present in the elution fraction from non-treated HeLa extract

(Figure 2). Our Phosphoprotein Enrichment Kit provided superior

and efﬁcient phosphoprotein enrichment yields when compared to

competitors’ products (Table 1). It also effectively enriched phos-

phoproteins from homogenized mouse liver tissue (Figure 3).

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch ofﬁce or distributor.

Table 1. The Thermo Scientiﬁc Pierce Phosphoprotein Enrichment Kit

provides higher phosphoprotein yields in less time than competitors’ kits.

Kit Yield (%) Enrichment Time (Hours)

Thermo Scientiﬁc Pierce

Phosphoprotein Enrichment Kit

15 1.5

Supplier Q Kit 4.4 4.5

Supplier I Kit 2.6* 3.5

Supplier C Kit 8 3

Supplier E Kit Too dilute to determine 5

*Based on maximum 1mg load per manufacturer’s protocol.

Phospho-MAPK T202/Y204

Phospho-PTEN S380

Phospho-GSK3β S9

Phospho-MAPK T202/Y204

Phospho-Akt S473

Phospho-Src Y527

Cytochrome C

p15Ink4b

NIH 3T3 + PDGF HeLa + EGF

FT W E L

Figure 1. Highly pure phosphoprotein enrichment from complex biological

samples. Serum-starved HeLa and NIH 3T3 cells were stimulated with EGF and

PDGF, respectively. Cell lysate (2mg) was used for enrichment. Concentrated

ﬂow-through, wash and elution fractions were resolved by SDS-PAGE. Gel

lanes were normalized by protein concentration (10μg/lane). Western blot

analysis was performed using antibodies that detect site-speciﬁc phosphory-

lation events. Cytochrome C (pI 9.6) and p15Ink4b (pI 5.5) served as negative

controls for nonspeciﬁc binding of non-phosphorylated proteins. FT = ﬂow-

through fraction, W = pooled wash fractions, E = pooled elution fractions and

L = non-enriched total cell lysate.

Phospho-PTEN S380

Phospho-MAPK T202/Y204

Phospho-GSK3β S9

Total Cell Lysate Elution

25 µg 10 µg 10 µg

λ Pase

- + - + - +

Figure 2. Highly specific phosphoprotein purification from lambda

phosphatase-treated cells. Non-treated and lambda dephosphorylated

HeLa cell extract (2mg) was loaded onto separate Thermo Scientiﬁc Pierce

Phosphoprotein Enrichment Columns. Concentrated elution fractions were

resolved by SDS-PAGE. Gel lanes were normalized by protein concentration

(10μg/lane). To determine enrichment, 10μg and 25μg of non-treated or lambda

phosphatase-treated total cell extract (non-enriched) was loaded onto each

gel. Western blot analysis was performed using phospho-speciﬁc antibodies

recognizing key proteins in the Ras-MAPK and PI3K-Akt signaling cascades.

L10 FT W E L25

Phospho-PTEN S380

Phospho-Rb S795

Cytochrome C

Figure 3. Efficient enrichment of phosphoproteins from mouse liver extract.

Homogenized mouse liver extract (~2mg) was loaded onto a Thermo Scientiﬁc

Pierce Phosphoprotein Enrichment Column. Concentrated ﬂow-through, wash

and elution fractions were resolved by SDS-PAGE. Gel lanes were normalized by

protein concentration (10μg/lane). Western blot analysis was performed using

antibodies that detect site-speciﬁc phosphorylation events. Cytochrome C

(pI 9.6) served as a negative control for nonspeciﬁc binding. L10 = non-enriched

total cell extract (10μg), FT = ﬂow-through fraction, W = wash fraction,

E = elution fraction and L25 = non-enriched total cell extract (25μg).

Ordering Information

Product #

Description

Pkg. Size

U.S.

Price

90003

Pierce Phosphoprotein Enrichment Kit

Includes: Phosphoprotein Enrichment Column

Resin Bed (1mL)

Lysis/Binding/Wash Buffer

Elution Buffer

CHAPS

White Column Caps

Pierce Protein Concentrator

7mL/9K MWCO

Kit

10 ea.

325mL

60mL

10 caps

10 Devices

$464

For more information, or to download product instructions, visit www.www.thermoscientiﬁc.com/pierce

Phosphoprotein Pull-Down with SH2 Domains

SH2 Domain Phosphotyrosine Capture Kits

SH2 domains provide an improved approach to selectively monitor

receptor tyrosine kinase signaling, as well as the binding of down-

stream effector proteins. The interactions of SH2 domain-contain-

ing proteins represent a critical interface between extracellular

stimulation of a membrane receptor and transmission of that

signal to intracellular proteins.

Our kits include optimal levels of puriﬁed GST-fused SH2 domains

of various signaling proteins integral to cell biology. Each validated

kit includes optimized buffers and columns to perform protein

pull-downs. Using SH2 domains eliminates the background

associated with low-speciﬁcity antibodies and enables analysis

of receptor targets to which antibodies are not available. Using

quality-tested puriﬁed GST-SH2 domains also ensures uniform

results without variability.

SH2 domains speciﬁcally bind phospho-tyrosine residues.

Each SH2 domain is speciﬁc for its natural target (Figure 4).

In vivo, the phosphatase Shp2 is recruited to tyrosine 1009 of the

platelet-derived growth factor receptor (PDGFR) only when it is

phosphorylated. Also, PLCg is recruited to tyrosine 1021 on PDGFR

in response to growth factor signaling. In quiescent cells both

tyrosine 1009 and 1021 are not phosphorylated and, therefore, both

domains are unable to bind.

PDGF

Antibody

GST-Shp2 GST-PLCγ

- +

pPDGFR Y1009 pPDGFR Y1021

Figure 4. Site-specific interaction of GST-Shp2 and GST-PLCg SH2 on the

PDGF-receptor. NIH3T3 cells were rendered quiescent by serum withdrawal

for 48 hours followed by stimulation with PDGF (50ng/mL, Cell Signaling

Technology [CST]) for 15 minutes or no stimulation. Each cell lysate (500μg) was

incubated overnight at 4°C with either 100μg GST-PLCg1 or GST-Shp2. Protein

complexes were captured on immobilized glutathione beads and resolved

by SDS-PAGE. Western blot analysis was performed using phospho-speciﬁc

antibodies that detect known protein-protein interaction sequences (phospho-

PDGFR Y1009 and phospho-PDGFR Y1021, CST). Lanes: L = 25μg of total cell

lysate and PD = SH2 domain pull-down.

SH2

Receptor in cell membrane

Extracellular signal

Tyrosine phosphorylation

SH2 domains recognize pY

Other proteins recruited

Translation, Transcription, Cell Proliferation, Differentiation, Apoptosis

Cellular pathway activation

SH2 domains provided a more efﬁcient method of capturing

site-speciﬁc phospho-tyrosine events as compared to antibody

based co-immunoprecipitation.

Co-immunoprecipitation experiments are dependent on the

quality and speciﬁcity of the antibody used. Antibodies against

phosphoproteins are notoriously difﬁcult to work with, yielding

high background and low target protein recovery. SH2 domains

provide better capture efﬁciency as compared to many pan- and

site-speciﬁc phospho-antibodies because an SH2 domain is more

selective by nature. Because of this improved speciﬁcity, SH2

domains can be used to pull-down phosphotyrosine containing

proteins with improved results over traditional antibody-based

methods. In Figure 5, PDGF-stimulated NIH3T3 cells were lysed

and incubated with either a speciﬁc SH2 domain or a pan-antibody

corresponding to the SH2 domain containing protein. Binding of

each speciﬁc SH2 domain to the PDGF receptor was conﬁrmed

by Western blot using a pan-PDGFR antibody. In all cases we

observed a higher capture efﬁciency using the SH2 domain

approach as compared to the traditional antibody co-immunopre-

cipitation. In many cases, we were also able to detect differential

binding of the SH2 domain in the presence of PDGF stimulation as

compared to the unstimulated state. These differences were not

apparent using the co-immunoprecipitation approach. We further

tested the interaction speciﬁcity of four SH2 domains using

phospho-antibodies which detect site-speciﬁc tyrosine

phosphorylation events which mediate each SH2 domain

interaction. We were limited to only four phospho-antibodies

against speciﬁc tyrosine sites on the PDGF receptor because

of commercial availability and/or antibody integrity. Using these

site-speciﬁc antibodies we conﬁrmed the selectivity and speciﬁcity

of each SH2 domain tested and these interactions were much

stronger as compared to the traditional antibody immunoprecipi-

tation. Taken together these results clearly demonstrate that the

SH2 domain pull-down approach represents a robust method to

capture and monitor site speciﬁc tyrosine phosphorylation events

which are critical for cell health.

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch ofﬁce or distributor.

Figure 5. SH2 domains provide better enrichment of phosphorylated

receptor tyrosine kinase (PDGFR) compared to antibody-based pull-down

assays. Panel A. NIH3T3 cells were rendered quiescent by serum starvation

for 24 hours with DMEM containing 0.1% FCS. Following starvation cells

were either stimulated (100ng/mL PDGF for 20 minutes) or left unstimulated.

Cells were then lysed in NP40 lysis buffer (50mm Tris pH 7.5, 150mm NaCl,

1% NP40, 5% glycerol) containing Thermo Scientiﬁc Halt Protease and

Phosphatase Inhibitor Cocktail (Product # 78440) and protein concentra-

tions determined by the Thermo Scientiﬁc Pierce 660nm Assay. SH2 and

co-immunoprecipitations were performed as follows: SH2 domain pull-downs

consisted of 100μg of SH2 domain (Abl, Crk, Grb2, Lyn, PI3K, Plc-gamma,

RasGap, Shc, Shp2, Src) added to 250μg of stimulated or unstimulated lysate.

Co-immunoprecipitations were performed by incubating 10μL of pan antibody

(Abl, Crk, Grb2, Lyn, PI3K, Plc-gamma, RasGap, Shc, Shp2, Src) with 250μg of

stimulated or non-stimulated lysate. Binding was performed for 16 hours at 4°C

on a rotating platform. Following binding 100μL of a 50% slurry of immobilized

glutathione was added to the SH2 domain samples and 100μL of a 50% slurry of

Protein A/G was added to the co-immunoprecipitation samples. Samples were

incubated for 1 hour at 4°C on a rocking platform followed by centrifugation at

1000 x g for 1 minute to collect complexes bound to the resin. Resin beds were

then washed 3 times with 500μL of Thermo Scientiﬁc M-PER Wash Buffer. To

elute bound proteins 25μL of 5X DTT loading dye was added to each sample

and boiled for 5 minutes. Protein complexes were then resolved by SDS-PAGE

on a 4-20% Tris-glycine gel. Proteins were transferred onto PVDF membrane

for 16 hours at 4°C, 30V. Membranes were blocked in 7.5% BSA in TBST and

Western blots performed using a 1:2000 dilution of mouse monoclonal

anti-PDGFR-beta antibody (CST 3175). Detection was performed using

Thermo Scientiﬁc SuperSignal Pico Chemilumescent Detection System.

Panel B. Membranes containing the Grb2, Plcg, PI3K, Src, and RasGap

pull-downs were stripped with Thermo Scientiﬁc Restore Plus Buffer and

reprobed with phospho-antibodies which detect site-speciﬁc tyrosine phos-

phorylation events on the PDGF receptor which mediate the SH2 domain

interaction. These antibodies include pPDGFRY716, pPDGFRY751,

pPDGFRY1009, pPDGFRY579 which correspond to SH2 domain docking sites

for Grb2, PI3K, Plcg, and Src respectively. Detection was performed using

SuperSignal

West Pico Chemilumescent Detection System.

PDGF

- + - + - + - + - + - + - + - + - + - +

PDGF

- + - + - + - + - + - + - + - + - + - +

SH2 IP SH2 IP SH2 IP SH2 IP SH2 IP

Abl Crk Grb2 Lyn PI3K

Plcγ

RasGap Shc Shp2 Src

Panel A.

Panel B.

Western Blot: Pan PDGFR

PDGFR

SH2: Grb2

IP: Pan Grb2

WB: pPDGFRY716

SH2: PI3K

IP: Pan PI3K

WB: pPDGFRY751

SH2: Plcγ

IP: Pan Plcγ

WB: pPDGFRY1009

SH2: Src

IP: Pan Src

WB: pPDGFRY579

PDGF

- + - + - + - + - + - + - + - +

SH2 IP SH2 IP SH2 IP SH2 IP

pPDGFR

For more information, or to download product instructions, visit www.www.thermoscientiﬁc.com/pierce

Perform receptor phosphorylation time-course experiments.

With SH2 domain pull-downs, it is possible to monitor time-dependent

binding of SH2 domains to their target. For example, PLCg, Src

and Shc bind rapidly to the PDGF receptor in response to PDGF

stimulation (Figure 6). In nature, phosphorylation is rapid and tran-

sient, as evidenced by diminished binding at 10- and 20-minute

post-stimulation.

Minutes

Post PDGF

Stimulation

GST-PLCγ

GST-Src

GST-Shc

0 1 5 10 20

Figure 6. Time-speciﬁc binding of various SH2 domains to the PDGF receptor.

NIH3T3 cells were rendered quiescent by serum withdrawal for 48 hours. Post-

starvation cells were stimulated with PDGF (50ng/mL, CST) for the indicated

times or untreated. SH2 domain pull-downs were performed with 100μg of each

SH2 domain and 250μg of each cell lysate. Protein complexes were resolved

by SDS-PAGE and analyzed by Western blot using a pan antibody recognizing

PDGFR (CST).

Resolve speciﬁc tyrosine phosphorylation events.

SH2 domain afﬁnity provides a level of selectivity not previously

attainable with generic anti-phosphotyrosine antibodies. Use

different SH2 domains to uncover the mechanism of how signals

are transduced or to screen multiple SH2 domains against a

particular target at one time. For example, different naturally

occurring SH2 domains efﬁciently bind to the epidermal growth

factor receptor (EGFR) in response to EGF stimulation (Figure 7).

There were strong interactions of many of the domains in the

presence of epidermal growth factor (EGF) and low-level binding

in quiescent, serum-starved A431 cells. RasGap binding is

independent of EGF stimulation, whereas Grb2 binding is highly

upregulated (Figure 7).

Ordering Information

Product #

Description

Pkg. Size

U.S.

Price

87700 Grb2 SH2 Domain Phosphotyrosine Capture Kit

Sufﬁcient reagents for six pull-down reactions

(100µg/pull-down).

Includes: GST-Grb2 SH2 Domain (37 kDa)

GST (negative control, 27 kDa)

Immobilized Glutathione Resin

(50% resin slurry)

Spin Column (0.8mL)

M-PER Mammalian Protein

Extraction Reagent

Lane Marker Reducing Sample Buffer (5X)

6 pull-downs

600μg at 1mg/mL

200μg at 1mg/mL

400μL

8 each

25mL

200μL

$408

87701 Src SH2 Domain Phosphotyrosine Capture Kit

6 pull-downs

$408

87702 Abl SH2 Domain Phosphotyrosine Capture Kit

6 pull-downs

$408

87703 Crk SH2 Domain Phosphotyrosine Capture Kit

6 pull-downs

$408

87704 Fyn SH2 Domain Phosphotyrosine Capture Kit

6 pull-downs

$408

87705 Lck SH2 Domain Phosphotyrosine Capture Kit

6 pull-downs

$408

L25

* * *

* **

EGF

+ – + – + – + – + – + –

EGF

* = Known Interactions

Antibody: Pan EGFR

** = GST Only: Negative Control

L25

+ –

Shp2

+ –

Grb2 Src PLCγ Hck Fyn

L25

+ – + – + – + – + – + –

Gap Abl Crk GST Nck

Figure 7. High efficiency binding of different naturally occurring SH2

domains to EGFR in response to EGF stimulation. A431 cells were rendered

quiescent by serum withdrawal for 48 hours followed by stimulation with EGF

(100ng/mL) for 15 minutes or left untreated. Each cell lysate (500μg) was incu-

bated with 100μg of each GST-SH2 domain overnight at 4˚C. Protein complexes

were captured on immobilized glutathione beads and resolved by SDS-PAGE.

Western blot analysis was performed using a pan antibody that recognizes

the EGF receptor. L25 = 25μg total lysate load.

Identify binding interactions with MS.

Pull-down samples isolated with SH2 domains are compatible with

MS analysis for identifying protein interacting partners.

Improved speciﬁcity with SH2 domains and the elimination of

antibody contamination typically seen in traditional immunopre-

cipitation experiments deliver pure protein for MS analysis. Two

different SH2 domains were used to pull down interacting proteins

from NIH3T3 cell lysates, which were then analyzed by MS. Many

of the interacting proteins identiﬁed are speciﬁc to either Fgr

or PLCg.

Ordering Information

Product #

Description

Pkg. Size

U.S.

Price

87706 Nck SH2 Domain Phosphotyrosine Capture Kit

6 pull-downs

$408

87707 Shc SH2 Domain Phosphotyrosine Capture Kit

6 pull-downs

$408

87708 Ras-GAP SH2 Domain Phosphotyrosine

Capture Kit

6 pull-downs

$408

87709 Shp2 SH2 Domain Phosphotyrosine Capture Kit

6 pull-downs

$408

87710 PLC SH2 Domain Phosphotyrosine Capture Kit

6 pull-downs

$408

87711 Lyn SH2 Domain Phosphotyrosine Capture Kit

6 pull-downs

$408

87712 Hck SH2 Domain Phosphotyrosine Capture Kit

6 pull-downs

$408

87713 FGR SH2 Domain Phosphotyrosine Capture Kit

6 pull-downs

$408

87714 Cbl SH2 Domain Phosphotyrosine Capture Kit

6 pull-downs

$408

87715 Syk SH2 Domain Phosphotyrosine Capture Kit

6 pull-downs

$408

87716 PI3K SH2 Domain Phosphotyrosine Capture Kit

6 pull-downs

$408

Phosphoprotein Pull-Down with SH2 Domains

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch ofﬁce or distributor.

Use of Titanium Dioxide for

Phosphopeptide Enrichment

Titanium dioxide enrichment of phosphopeptide samples is an

essential tool for comprehensive MS analysis of phosphopeptides.

The unique phosphopeptide binding properties of TiO

resin results

in different selectivity and preferential enrichment of multiply

phosphorylated peptides compared to immobilized chelated metal

ions (IMAC). TiO

resin typically enriches more phosphopeptides

than IMAC resins, and TiO

preferentially enriches monophos-

phorylated peptides.

TiO

resin typically binds phosphopeptides with a higher afﬁnity

than IMAC and the preferential enrichment of singly phosphory-

lated peptides with TiO

is likely due to difﬁculty in eluting multiply

phosphorylated peptides. To enrich a more comprehensive set of

phosphopeptides, the ﬂow-through and washes from IMAC resin

can be acidiﬁed and applied to TiO

resins and the resulting elu-

ates pooled for analysis. In addition, the number of phosphopep-

tides recovered can be signiﬁcantly increased by pre-fractionation

of samples with strong cation exchange chromatography to

reduce sample complexity prior to enrichment with TiO

or IMAC

processing.

Thermo Scientiﬁc Pierce TiO

Phosphopeptide Enrichment and

Clean-up Kit

Selective enrichment and Clean-up of phosphopeptides in under

2 hours.

The Pierce TiO

Phosphopeptide Enrichment and Clean-up Kit

enables fast, selective enrichment of phosphorylated peptides.

The complete kit includes 24 titanium dioxide (TiO

) spin tips and

graphite spin columns with buffers to facilitate preparation of

enriched and desalted phosphopeptides for analysis by mass

spectrometry (MS).

Highlights:

• Convenient – spin-column format of TiO

and graphite columns

enable parallel processing and clean up of multiple samples in

less than 2 hours

• High capacity – each column enriches up to 300μg of

phosphopeptides

• Complementary – TiO

enriches a unique set of phosphopeptides

that complements the Pierce Fe-NTA IMAC Phosphopeptide

Enrichment Kit

• High selectivity – recover phosphopeptides with >90% selectivity

The Pierce TiO

Phosphopeptide Enrichment and Clean-up Kit

protocol includes stringent washing conditions that increase the

selectivity for phosphopeptides to >95%. Because some of the

salts in these stringent washes may still be present in the eluted

samples, the Pierce TiO

Phosphopeptide Enrichment and Clean-

up Kit includes graphite spin columns to desalt and concentrate

enriched phosphopeptides, resulting in more successful phos-

phopeptide analysis results. This kit is compatible with samples

digested in solution or after in-gel digestion with trypsin or other

MS grade proteases. Using the Pierce TiO

Phosphopeptide

Enrichment and Clean-up Kit and the Pierce Fe-NTA IMAC

Phosphopeptide Enrichment Kits will enable complementary sets

of phosphopeptides to be removed from complex samples.

Number of Phosphates per Pepti0de

Resin

1 2 3 4 5 6

Total

TiO

492 103 8 4 0 1 608

Fe-NTA IMAC

234 34 216 3 1 0 488

Overlap

155 0 1 0 0 0 156

Selective enrichment of singly and multiply phosphorylated phosphopeptides

with Thermo Scientiﬁc Pierce TiO

and FE-NTA IMAC resins. Average phos-

phopeptide enrichment results from duplicate experiments showing the number

of phosphopeptides containing one or more phosphate per peptide enriched

using either resin. Peptide spectrum summary results were exported from

Proteome Software Scaffold 3.0.

120

100

Total Phosphopeptides

Unique Phosphopeptides

% Acidic Residues

TiO2 Fe-NTA

Phosphopeptides (%)

961

240

998

238

TiO

selectively enriches more phosphopeptides than Fe-NTA IMAC. The num-

bers refer to the total and unique number of phosphopeptides enriched by each

method. The Y-axis is the percentage of phosphopeptides in the number of total

and unique peptides, or the percentage of acidic residues in enriched unique

phosphopeptides.

References:

Larsen M.R., et al. (2005). Highly selective enrichment of phosphorylated peptides from

peptide mixtures using titanium dioxide microcolumns. Mol Cell Proteomics. 4(7):873-86.

Carrascal, M., et al. (2008). Phosphorylation Analysis of Primary Human T Lymphocytes

Using Sequential IMAC and Titanium Oxide Enrichment. J. Proteome Res. 7(12):5167-5176.

Wilson-Grady, J.T., et al. (2008). Phosphoproteome Analysis of Fission Yeast. J. Proteome

Res. 7(3):1088-1097.

Larsen, M.R., et al. (2004). Improved detection of hydrophilic phosphopeptides using

graphite powder microcolumns and mass spectrometry: evidence for in vivo doubly phos-

phorylated dynamin I and dynamin III. Mol. Cell Proteomics. 3:456-465.

Ordering Information

Product #

Description

Pkg. Size

U.S.

Price

88301 Pierce TiO

Phosphopeptide Enrichment

and Clean-up Kit

Sufﬁcient For: 24 phosphopeptide enrichment and

cleanup reactions.

Kit Contents: TiO

Spin Tips, 24 tips

Triﬂuoroacetic Acid, 1mL

90% Lactic Acid, 2mL

Pyrrolidine, 200μL

Centrifuge Column Adaptors, 24 adaptors

Pierce Graphite Spin Columns, 24 columns

24-Rxn Kit

$375

Phosphopeptide Enrichment

For more information, or to download product instructions, visit www.www.thermoscientiﬁc.com/pierce

Phosphopeptide Enrichment

Thermo Scientiﬁc Pierce Magnetic Titanium Dioxide

Phosphopeptide Enrichment Kit

TiO

magnetic particles for high throughput phosphopeptide isolation.

The Pierce Magnetic Titanium Dioxide Phosphopeptide Enrichment

Kit is for isolating phosphopeptides from complex biological sam-

ples using titanium dioxide-coated magnetic beads. The TiO

ligand

selectively binds peptides con-taining phosphorylated serine

(Ser), tyrosine (Tyr) or threonine (Thr), enabling phosphopeptide

enrichment from protease-digested samples. The isolated phos-

phopeptides are compatible for analysis downstream by mass

spectrometry (Table 2).

The high-performance, iron oxide, superparamagnetic particles

are validated and optimized for use with high-throughput

magnetic platforms, such as the Thermo Scientiﬁc KingFisher 96

and KingFisher

Flex Instruments. The beads also enable premium

performance for simple benchtop applications using an appropriate

magnetic stand.

Highlights:

• Complete MS-compatible Kits – include ready-to-use binding,

wash and elution buffers that are optimized for phosphopeptide

enrichment and downstream analysis by MALDI and ESI

mass spectrometry

• Optimized for HTS – procedure validated for processing 1 to 96

samples at a time; complete entire assay in about 15 minutes

using a KingFisher Flex Instrument

• Stable afﬁnity ligand – titanium dioxide is specially coated as a

ﬁlm on the magnetic particles

• Selective – afﬁnity system is selective for phosphorylated Ser, Tyr

and Thr; exhibits minimal non-speciﬁc binding to acidic residues

• Sensitive – afﬁnity provides more than 1000 times greater sensi-

tivity than traditional IMAC technologies; enables enrichment and

MS-measurement of less than 100fmol of phosphoprotein

Table 2. Phosphopeptide enrichment improves MS-identiﬁcation of phospho-

proteins. Two milligrams of a tryptic digest prepared from periferal blood mono-

nuclear cells (lymphocytes) with and without phosphopeptide enrichment were

analyzed by MS. Enrichment was performed with the Thermo Scientiﬁc Pierce

Titanium Dioxide Phosphopeptide Enrichment Kit using the Thermo Scientiﬁc

KingFisher 96 Instrument. Samples were analyzed on a Thermo Scientiﬁc LTQ

Orbitrap Mass Spectrometer.

Enriched Non-Enriched

Total number of proteins identiﬁed 185 247

Total number of phosphoproteins identiﬁed 160 1

Total number of peptides identiﬁed 2347 2457

Total number of phosphopeptides identiﬁed 2009 7

Total number of unique

phosphopeptides identiﬁed 177 1

Relative enrichment for phosphopeptides (%) 86 0.3

Ordering Information

Product #

Description

Pkg. Size

U.S.

Price

88811 Pierce Magnetic Titanium Dioxide

Phosphopeptide Enrichment Kit

Includes: TiO

Magnetic Beads (20X)

Binding Buffer

Washing Buffer

Elution Buffer

Thermo-Fast 96 Robotic PCR Plate

(0.2mL wells)

Kit

1mL

100mL

25mL

3mL

2 plates

$427

88812 Pierce Magnetic Titanium Dioxide

Phosphopeptide Enrichment Kit, Trial Size

Includes: TiO

Magnetic Beads (20X)

Binding Buffer

Washing Buffer

Elution Buffer

Thermo-Fast 96 Robotic PCR Plate

(0.2mL wells)

Kit

0.25mL

100mL

25mL

3mL

2 plates

$240

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch ofﬁce or distributor.

Thermo Scientiﬁc Pierce Fe-NTA Phosphopeptide Enrichment Kit

New Fe-NTA format optimized for capture and recovery

of phosphopeptides.

The new Pierce Fe-NTA Phosphopeptide Enrichment Kit enables

fast and efﬁcient enrichment of phosphorylated peptides. These

spin columns are easy to use and require less than 1 hour to

process protein digests or strong cation-exchange peptide

fractions for analysis by mass spectrometry (MS).

Highlights:

• Convenient spin format for parallel processing of

multiple samples

• High-binding capacity resin for enriching up to 150μg

of phosphopeptides per column

• Excellent enrichment and recovery of phosphopeptides

Protein phosphorylation is essential to biological functions,

including cell signaling, growth, differentiation, division and

programmed cell death. Over 500 protein kinases catalyze

phosphorylation of speciﬁc targets, primarily on serine, threonine,

and tyrosine residues.

Mass spectrometry is increasingly being used to identify and

quantify phosphorylation changes; however, phosphoprotein

and phosphopeptide analysis by MS is limited by many factors,

including digestion efﬁciency, low stoiochiometry, low abundance,

hydrophilicity, poor ionization and poor fragmentation. As a result,

phosphopeptide enrichment is essential to successful MS analy-

sis. The new Pierce Fe-NTA Phosphopeptide Enrichment Kit is

compatible with our lysis, reduction, alkylation, and digestion

reagents and with Thermo Scientiﬁc Pierce Graphite Spin Columns

to provide a complete workﬂow for phosphopeptide enrichment.

To assess phosphopeptide enrichment from lysates, cultured

U2-OS cells arrested with nocodazole (100ng/mL, 25 hours)

were lysed with 6M urea in 50mM Tris, pH 8.0 containing Thermo

Scientiﬁc Halt Protease and Phosphatase Inhibitor Cocktail

(Product # 78440). Protein concentration was determined with

Thermo Scientiﬁc Pierce 660nm Protein Assay (Product # 22660).

Proteins were reduced with Thermo Scientiﬁc Bond-Breaker

TCEP Solution, Neutral pH (Product # 77720), alkylated with single-

use iodoacetamide, (Product # 90034) digested overnight with

MS-grade trypsin (Product # 90055), and desalted with Thermo

Scientiﬁc HyperSep-C18 Cartridges (Product # 60108-305). An

equivalent of 200μg of peptides were dried and dissolved in 5%

acetic acid or Sigma Phos-Select

™

Buffer. Phosphopeptides were

enriched with Pierce Fe-NTA Phosphopeptide Enrichment Kit or

Sigma Phos-Select Reagents and then desalted and concentrated

with Pierce Graphite Spin Columns (Product # 88302) according

to instructions.

Enriched phosphopeptide samples were analyzed by LC-MS/MS.

A NanoLC

™

-2D HPLC (Eksigent) with a ProteoPep II C18 Column

(75μm ID x 20cm, New Objective) was used to separate peptides

using a 4-40% gradient of solvents (A: water, 0.1% formic acid;

B: acetonitrile, 0.1% formic acid) at 250nL per minute for 60

minutes. Peptides were identiﬁed with a Thermo Scientiﬁc LTQ

Orbitrap XL ETD Mass Spectrometer using a top four experiment

consisting of high-resolution MS followed by acquisition of four

MS/MS spectra using the CID mode of fragmentation. LC-MS/

MS data were interpreted with Mascot 2.2 (Matrix Science) and

Scaffold 2.6 (Proteome Software).

To achieve robust MS results, enrichment of phosphopeptide

samples is essential because of low stoichiometry and abundance

and poor ionization relative to nonphosphorylated peptides. We

have developed an efﬁcient means to enrich phosphopeptides

from complex samples. The new Thermo Scientiﬁc Pierce

Fe-NTA Spin Columns effectively capture, enrich, and recover

phosphopeptides. These columns enrich a higher percentage of

phosphopeptides than other resins and with an overall higher

number of total and unique phosphopeptides (Figure 8

and Table 3).

Phosphopeptides (%)

Thermo Scientific Pierce Fe-NTA Kit Sigma Phos-Select

Reagent

Total Phosphopeptides

862

430

178

Unique Phosphopeptides

Figure 8. Our kit enriched a greater percentage of total and unique phospho-

peptides from U2-OS cell lysate. Numbers refer to the total and unique num-

ber of phosphopeptides identiﬁed in each condition. A summary of

results is listed in Table 3.

For more information, or to download product instructions, visit www.www.thermoscientiﬁc.com/pierce

Table 3. Average phosphopeptide enrichment results from duplicate experiments.

Thermo Scientiﬁc

Pierce Fe-NTA

Sigma Phos-Select

Total phosphopeptides 862 430

Total peptides 1,753 1,665

Total unique peptides 393 395

Total unique phosphopeptides 178 90

Total phosphopeptides (%) 53 31

Unique phosphopeptides (%) 50 27.5

§ Peptide summary results were exported from Scaffold and analyzed and summarized

with Microsoft Excel

and Access

Multiple phosphorylated amino acids within a peptide contribute

to the complexity of phosphopeptide analysis. Pierce Fe-NTA Spin

Columns enrich peptides with three or more phosphorylated sites

and signiﬁcantly outperform other commercially available columns

(Figure 9). Phosphopeptide enrichment greatly reduces sample

complexity and enables effective identiﬁcation and characteriza-

tion of phosphorylated peptides by MS (Figure 10).

100

120

Phosphopeptides (%)

Single Double

Phosphates per Peptide

Triple ≥4

Thermo Scientific Pierce Fe-NTA Kit

110

12 1 0

Sigma Phos-Select Reagent

Figure 9. The Thermo Scientiﬁc Pierce Fe-NTA Phosphopeptide Enrichment Kit

effectively captures phosphopeptides with multiple phosphates.

The Pierce Fe-NTA Phosphopeptide Enrichment Kit contains

detailed instructions and all necessary components to load, wash

and elute phosphopeptides within an hour. This kit is compatible

with samples digested in solution or after in-gel digestion using the

Thermo Scientiﬁc In-gel Tryptic Digestion Kit (Product # 89871).

a11+2H+1

y2 b4

b9 y9

b10

G E P N

V S Y+80 I C+57 S R

R S

C+57 I Y+80 S V N P

E G

m/z

25%

50%

75%

100%

Relative Intensity

0 250 500 750 1000 1250

m/z

25%

50%

75%

100%

Relative Intensity

0 250 500 750 1000 1250

GSK3A_HUMAN

Glycogen Synthase Kinase-3 alpha

(R)GEPNVSyICSR(Y)

CDC2_HUMAN

Cell division control protein 2 homolog

(K)IGEGTyGVVYK(G)

b10

y10

I G E

G T Y+80 G V V Y K

K Y

V V G Y+80 T G E G I

Ordering Information

Product #

Description

Pkg. Size

U.S.

Price

88300 Pierce Fe-NTA Phosphopeptide

Enrichment Kit

Sufﬁcient for 30 samples

Kit

$279

Related Products

88302 Pierce Graphite Spin Columns

30 columns

$105

Figure 10. Example MS/MS

spectra from enriched

phosphopeptides. Lowercase

letters indicate the position

of tyrosine phosphorylation

in enriched peptides.

Panel A: Glycogen synthase-3

alpha. Panel B: cdc2/cyclin

dependent kinase 1.

Phosphopeptide Enrichment

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch ofﬁce or distributor.

Cell Surface Protein Isolation Kit

Convenient biotinylation and isolation of cell surface proteins

for Western blot analysis.

The Thermo Scientiﬁc Pierce Cell Surface Protein Isolation Kit

is a complete kit for the convenient biotinylation and isolation

of mammalian cell surface proteins, speciﬁcally targeting cell

surface proteins to the exclusion of intracellular proteins. The

kit efﬁciently labels proteins with accessible lysine residues and

sufﬁcient extracellular exposure.

The isolation procedure uses a cell-impermeable, cleavable

biotinylation reagent (Sulfo-NHS-SS-Biotin) to label surface

proteins at exposed primary amines. Cells are then harvested and

lysed, and the labeled surface proteins are afﬁnity-puriﬁed using

Thermo Scientiﬁc NeutrAvidin Agarose Resin. The isolated cell

surface proteins contain a small, nonreactive tag of the originally

labeled primary amines but are no longer biotinylated (biotin

remains bound to the resin).

Highlights:

• Isolates cell surface proteins – reduces complexity of total

cellular protein

• Efﬁciently recovers labeled proteins – cleavable biotin allows for

nearly 100% recovery of isolated cell surface proteins

• Convenience – all reagents are provided in one kit, along with

complete instructions for labeling, cell lysis and puriﬁcation of

cell surface membrane proteins

• Western blotting applications – proteins recovered in SDS-PAGE

buffer are loaded directly onto polyacrylamide gels

• Robust system – protocol designed for diverse cell lines,

including NIH 3T3, HeLa, C6 and A431

A. Cell Surface Proteins

EGFR

IGF-1Rβ

Hsp90

Calnexin

Integrin α5

Integrin β1

R F E R F E

R F E R F E R F E R F E

R F E R F E

B. Intracellular Proteins

+ – + –

+ –

Protein isolation is specific to cell surface proteins. Panels are Western blot

results for known cell surface proteins (Panel A) and intracellular proteins

(Panel B) from HeLa cells tested with the Cell Surface Protein Isolation Kit.

Plus symbol (+) denotes results for cells treated with the Sulfo-NHS-SS-Biotin

reagent; minus symbol (-) denotes results for cells that were not treated with

the biotin reagent but were otherwise carried through the kit procedure.

Lanes are no-sample resin-control (R), flow-through (F) and eluted (E) frac-

tions. Presence of target cell surface proteins in the plus-E and minus-F

conditions indicate successful isolation with the kit. Presence of intracellular

proteins in F condition of both plus and minus conditions indicates that the

labeling and purification procedure is specific to cell surface proteins.

Ordering Information

Product # Description Pkg. Size

U.S.

Price

89881 Cell Surface Protein Isolation Kit

Sufﬁcient reagents and accessories for eight

experiments, each involving four T75 ﬂasks of

conﬂuent cells.

Includes: EZ-Link

Sulfo-NHS-SS-Biotin

Quenching Solution

Lysis Buffer

NeutrAvidin Agarose

Wash Buffer

Dithiothreitol (DTT)

BupH Phosphate Buffered Saline

BupH Tris Buffered Saline

Spin Columns and Accessories

Kit

8 x 12mg vials

16mL

4.5mL

2.25mL

34mL

8 x 7.7mg

microtubes

2 packs

1 pack

$348

Biotinylate cells

30 minutes at 4˚C

Transfer cell pellet

to 1.5 ml tube

Lyse cells

30 minutes on ice

Isolate biotinylated proteins

on NeutrAvidin

Agarose Resin

Perform

electrophoresis

or other application

Wash gel then elute with SDS-PAGE

sample buffer + 50 mM DTT

Harvest Cells

Quench Reaction

gel

protein – SH

gel

S-S-protein

Biotin

NeutrAvidin

Biotin-Binding Protein

1D gel

7.5

5.0

Protocol summary for the Thermo Scientiﬁc Pierce Cell Surface Protein Isolation Kit.

Protein Enrichment

For more information, or to download product instructions, visit www.www.thermoscientiﬁc.com/pierce

Glycoprotein Isolation Kits

Isolate glycoproteins from complex protein mixtures.

Two lectin-based Thermo Scientiﬁc Glycoprotein Isolation Kits,

concanavalin A (ConA) and wheat germ agglutinin (WGA), allow

isolation of glycoproteins from complex protein mixtures, including

serum, tissue and cultured cell lysates, thus enabling downstream

analysis. ConA lectin recognizes a-linked mannose and terminal

glucose residues, while WGA lectin selectively binds to N-acetyl

glucosamine (GlcNAc) groups and to sialic acid.

Highlights:

• High recovery – equivalent or greater glycoprotein recovery vs.

competitor kits and lectin resins

• Fast – glycoprotein puriﬁcation in less than one hour

• Versatile – isolate glycoproteins from various sample types;

e.g., human serum and cell lysate

• Robust – lectin does not leach from resin when

processing sample

• Convenient – complete kit contains lectin resins and spin

columns with all necessary reagents

• Compatible with Bradford-based protein assays – dialysis or

protein precipitation of recovered glycoproteins is not required

before protein assay

Thermo Scientific

Supplier A

Supplier G

ConA

Supplier G

ConA

Human Serum CHO Lysate

Glycoprotein isolation from human serum and cell lysate: performance

comparison of kits using ConA resin. Human serum and CHO lysate samples

were processed with the Thermo Scientiﬁc Glycoprotein Isolation Kit, ConA

and with other commercially available ConA resins. An equivalent amount of

total protein was applied to each resin. Eluted glycoprotein fractions were

compared with ConA Resin boiled in SDS-PAGE Buffer to release lectins. All

fractions were normalized by volume and resolved on 8-16% polyacrylamide

gels. Gels were silver-stained. A. Eluted glycoprotein fractions from applied

human serum and B. eluted glycoprotein fractions from applied CHO lysate.

Arrows identify protein bands that result from ConA leaching from the resin

during the elution process.

Competitor A

Supplier A

CHO LysateHuman Serum

Thermo Scientific

Glycoprotein isolation from human serum and cell lysate: performance

comparison of kits using WGA resin. Human serum and CHO lysate samples

were processed with the Thermo Scientiﬁc Glycoprotein Isolation Kit, WGA

and with other commercially available WGA resins. An equivalent amount of

total protein was applied to each resin. Eluted glycoprotein fractions were

normalized by volume and resolved on 8-16% polyacrylamide gels. A. Eluted

glycoprotein fraction from applied human serum and B. eluted glycoprotein

fraction from applied CHO lysate.

Ordering Information

Product #

Description

Pkg. Size

U.S.

Price

89804

Glycoprotein Isolation Kit, ConA

Sufﬁcient reagents to isolate glycoproteins with

strong afﬁnity for ConA from 10 samples of up to 640µL

(1-1.5mg total protein) each.

Includes: ConA Lectin Resin, 1.1mL resin supplied as

a 50% slurry

Binding/Wash Buffer, 5X Stock Solution

Elution Buffer

Column Accessory Pack, 10 Spin Columns

with Caps and 20 Collection Tubes

Kit

6.5mL

5mL

$223

89805 Glycoprotein Isolation Kit, WGA

Sufﬁcient reagents to isolate glycoproteins with strong

afﬁnity for WGA from 10 samples of up to 640µL

(1-1.5mg total protein) each.

Includes: WGA Lectin Resin, 1.1mL resin supplied as a

50% slurry

Binding/Wash Buffer, 5X Stock Solution

Elution Buffer

Column Accessory Pack, 10 Spin Columns

with Caps and 20 Collection Tubes

Kit

6.5mL

5mL

$282

Protein Enrichment

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch ofﬁce or distributor.

Ubiquitin Enrichment Kit

Recover ubiquitin modiﬁed protein in <45 minutes.

The ubiquitin proteasome pathway is the principal non-lysosomal

pathway that controls the proteolysis of proteins. This pathway

is signiﬁcantly involved in a variety of cellular processes, including

DNA repair, transcriptional regulation, signal transduction, cell

metabolism and morphogenesis. Differences in total ubiquitination

or the ubiquitination of speciﬁc proteins affect numerous pathological

conditions, including malignancies, certain genetic diseases and

neurodegenerative diseases.

The Thermo Scientiﬁc Ubiquitin Enrichment Kit isolates

polyubiquitin protein conjugates from cultured cells and tissue

samples. The enriched fraction is analyzed to determine the

amount of general ubiquitin conjugates present or to identify a

speciﬁc protein by Western blotting. The assay protocol is fast,

straightforward and allows isolation of polyubiquitinated proteins

and the fractionation of monoubiquitinated species in the resin

ﬂow-through. The Ubiquitin Enrichment Kit outperforms other

suppliers’ kits and provides a clean and speciﬁc preparation of

proteins when compared to a control resin.

Highlights:

• Fast – less than 45 minutes hands-on time

• Complete – includes all reagents needed for ubiquitin-modiﬁed

protein enrichment from cultured cells and tissue samples,

including spin columns and ubiquitin antibody

• Flexible – sample incubation from 2 hours to overnight allows

assay to be completed in several hours or in less than 30 minutes

after overnight incubation

• Robust – compatible with all Thermo Scientiﬁc Cell Lysis

Solutions and standard RIPA formulations

• Multiple-sample format – easily processes 1-15 samples

concurrently

100

120

220

Thermo

Scientific

Kit

Supplier

Ab-based

Method

GSH

Resin

H F E F E F E F E

kDa

The Thermo Scientific Ubiquitin Enrichment Kit recovers more ubiquitin-

modified proteins than any other method. Epoxomicin-treated HeLa cell

lysates (EHeLa, 150μg) were enriched. After elution, all samples were

normalized to the initial load (EHeLa load). The ﬂow-through and elution

obtained using the Thermo Scientiﬁc Ubiquitin Enrichment Kit are shown ﬁrst.

The results using another supplier’s enrichment kit are shown for comparison

(Supplier C, manufacturer’s instructions for this kit were followed). Additionally,

the results obtained using an anti-ubiquitin monoclonal antibody-based

enrichment scheme (antibody-based) and a negative control resin (GSH resin)

are shown. The elution from each resin shows the amount of ubiquitin-modiﬁed

protein that was captured using that method.

Reference

1. Ciechanover, A. (1998). The ubiquitin-proteasome pathway: on protein death and cell life.

EMBO J. 17(24), 7151-1760.

Ordering Information

Product # Description Pkg. Size

U.S.

Price

89899

Ubiquitin Enrichment Kit

Contains sufﬁcient materials for enriching up to

15 lysate samples containing ~0.15mg total protein

per sample.

Pack 1

Polyubiquitin Positive Control (1,000X), 50μL, 2mg/mL

Anti-ubiquitin Antibody, 50μL rabbit antiserum

Pack 2

Polyubiquitin Afﬁnity Resin, 300μL, supplied as a

25% slurry

Binding Capacity: ~1μg per 20μL of slurry

BupH Tris Buffered Saline Pack, 1 ea., makes

500mL of 0.025M Tris, 0.15M NaCl; pH 7.2

Spin Columns and Accessories, 18 columns with top

and bottom caps

Kit

$399

For more information, or to download product instructions, visit www.thermoscientiﬁc.com/pierce

Antibody Puriﬁcation Overview

Antibodies speciﬁc for an antigen of interest are one of the most

useful and important tools that biology researchers can possess.

The production and use of speciﬁc antibodies as detection

probes and puriﬁcation ligands (i.e., immunotechnology) has

revolutionized bioresearch and diagnostic technologies. Animals

immunized with prepared antigens will produce speciﬁc antibodies

against the antigen. When puriﬁed from serum or hybridoma cell

lines that are prepared from tissue of the immunized animal, the

antibody can be used directly (or after labeling with enzyme or

ﬂuorescent tags) to probe the speciﬁc antigen in Western blotting,

ELISA or a variety of other applications. Antibodies are most

commonly puriﬁed by one of two afﬁnity puriﬁcation methods:

general immunoglobulin puriﬁcation (pages 58-65) or speciﬁc

antibody puriﬁcation (see pages 26-35). See also Table 1.

General Puriﬁcation of Immunoglobulins

Because antibodies have predictable structure, including relatively

invariant domains, it has been possible to identify certain protein

ligands that are capable of binding generally to antibodies,

regardless of the antibody’s speciﬁcity to antigen. Protein A,

Protein G and Protein L are three bacterial proteins whose

antibody-binding properties have been well characterized. These

proteins have been produced recombinantly and used routinely for

afﬁnity puriﬁcation of key antibody types from a variety of species.

A genetically engineered recombinant form of Protein A and G,

called Protein A/G, is also available. These antibody-binding

proteins are available immobilized to beaded agarose resin,

Thermo Scientiﬁc UltraLink Biosupport and coated onto microplates.

Proteins A, G, A/G and L bind to antibodies at sites other than the

antigen-binding domain. Therefore, these proteins can be used in

puriﬁcation schemes such as immunoprecipitation.

Proteins A, G, A/G and L have unique properties, which make each

one suitable for different types of antibody targets (e.g., antibody

subclass or animal species). It is important to realize that use of

Protein A, G or L results in puriﬁcation of general immunoglobulin

from a crude sample. Depending on the sample source, antigen-

speciﬁc antibody may account for only a small portion of the total

immunoglobulin in the sample. For example, generally only 2-5%

of total IgG in mouse serum is speciﬁc for the antigen used to

immunize the animal.

Table 1. Antibody Puriﬁcation Methods.

Puriﬁcation Type Description Available Thermo Scientiﬁc Support

General Negative selection Removal of all non-immunoglobulin proteins from a

serum sample

Melon Gel

IgG enrichment Immobilized globulin binding proteins to selectively

remove IgG from a serum sample

Immobilized Protein A

Immobilized Protein G

Immobilized Protein A/G

Immobilized Protein L

IgG enrichment Thiophilic adsorption Thiophilic Adsorbent

IgM enrichment Use Mannan binding protein to selectively isolate IgM Immobilized Mannan Binding Protein

IgA enrichment Use Jacalin, a D-galactose binding lectin, to selectively

isolate IgA

Immobilized Jacalin

IgY enrichment Delipidation and precipitation from egg yolks IgY Precipitation Reagent

Speciﬁc

(see page 26)

Afﬁnity puriﬁcation Create an afﬁnity column with the antigen used to

immunize the animal.

NHS-ester Activated Agarose - for immobilizing antigens via

primary amines

AminoLink Plus Agarose - for immobilizing antigens via primary

amines

SulfoLink Resin - for immobilizing antigens via reduced sulfhydryls

CarboxyLink Resin - for immobilizing antigens via carboxylic acids

Antibody Puriﬁcation

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch ofﬁce or distributor.

w = weak binding, m = medium binding, s = strong binding, nb = no binding, – means information not available

* Data represent a summary of binding properties reported in the literature. Inevitably some discrepancies exist among reported values as a result of differences in binding

buffer conditions and form of the proteins used.

†

Binding will occur only if the appropriate kappa light chains are present. Antibodies lambda light chains will not bind, regardless of their class and subclass.

Protein A Protein G Protein A/G Protein L

†

Thiophilic

Adsorbent

Human IgG s s s s m

Mouse IgG s s s s s

Rabbit IgG s s s w m

Goat IgG w s s nb s

Rat IgG w m m s s

Sheep IgG w s s nb s

Cow IgG w s s nb s

Guinea Pig IgG s w s – s

Hamster IgG m m m s –

Pig IgG s w s s s

Horse IgG w s s – s

Donkey IgG m s s – –

Dog IgG s w s – s

Cat IgG s w s – s

Monkey IgG

(Rhesus)

s s s – s

Chicken IgY nb nb nb nb m

Human IgM w nb w s m

Human IgE m nb m s –

Human IgD nb nb nb s –

Human IgA w nb w s m

Human IgA

w nb w s m

Human IgA

w nb w s m

Human IgG

s s s s m

Protein A Protein G Protein A/G Protein L

†

Thiophilic

Adsorbent

Human IgG

s s s s m

Human IgG

w s s s m

Human IgG

s s s s m

Human Fab w w w s m

Human ScFv w nb w s m

Mouse IgG

w m m s s

Mouse IgG

s s s s s

Mouse IgG

s s s s s

Mouse IgG

s s s s s

Rat IgG

w m m s s

Rat IgG

nb s s s s

Rat IgG

nb w w s s

Rat IgG

s s s s s

Cow IgG

w s s nb s

Cow IgG

s s s nb s

Sheep IgG

w s s nb s

Sheep IgG

s s s nb s

Goat IgG

w s s nb s

Goat IgG

s s s nb s

Horse IgG(ab) w nb w – s

Horse IgG(c) w nb w – s

Horse IgG(T) nb s s – s

Mouse IgM nb nb nb s m

Immobilized Protein L, Protein A, Protein G

and Protein A/G

We offer these popular antibody-binding proteins immobilized on

several different resins, beads and plates for use in immunoafﬁnity

puriﬁcation techniques. All four proteins (A, G, A/G and L) are

available as puriﬁed recombinants immobilized to crosslinked 6%

beaded agarose. This is the traditional format historically used for

small-scale column puriﬁcation and immunoprecipitation methods.

Our agarose resins differ from those typically offered by other

suppliers in that our immobilization method is more stable and

results in less nonspeciﬁc binding. We also offer “Plus” versions

of the Protein A, G, A/G and L agarose resins, which contain twice

the amount of protein per milliliter of resin and provide for nearly

twice the antibody binding capacity.

Protein A, G, A/G and L are also available immobilized to UltraLink

Biosupport, an extremely durable, polyacrylamide-based resin with

very low nonspeciﬁc binding characteristics. The UltraLink Format

is a perfect support for working with large volume samples in large-

scale puriﬁcation methods requiring fast ﬂow and high pressure.

The interaction between the various proteins and IgG is not

equivalent for all species or all antibody subclasses. The tables

on the following page will help you decide which afﬁnity protein is

best for your application (Tables 2 and 3).

Table 2. Characteristics of immunoglobulin-binding proteins.

Recombinant

Protein L

Native

Protein A

Recombinant

Protein A

Recombinant

Protein G

Recombinant

Protein A/G

Production Source E. coli S. aureus E. coli E. coli E. coli

Molecular Weight 35,800 46,700 44,600 21,600 50,460

Number of Binding Sites for IgG 4 4 4 2 6

Albumin-Binding Site No No No No No

Optimal Binding pH 7.5 8.2 8.2 5 5-8.2

Binds to V

K F

Table 3. Binding characteristics of immunoglobulin-binding proteins and Thermo Scientiﬁc Thiophilic Adsorbent.*

For more information, or to download product instructions, visit www.thermoscientiﬁc.com/pierce

Protein A

Protein A Beads – Quick Reference

Protein A Native protein puriﬁed from

Staphylococcus aureus

(46.7kDa; four IgG-binding sites)

Speciﬁcity

(Table 3)

Best for polyclonal IgG from rabbit, pig, dog, cat serum;

poor for Mouse IgG1, human IgG3, rat, goat, cow

Supports

Offered

Crosslinked 6% beaded agarose

UltraLink Biosupport

Trisacryl GF-2000 Resin

Magnetic particles (1-4μm)

Package

Formats

Resin slurries (3 sizes)

Chromatography Cartridges (2 sizes)

Packed Spin Columns (3 sizes)

NAb

™

IgG Puriﬁcation Kits (2 sizes)

Storage 4°C, do not freeze

Capacity Protein A: 12-19mg human IgG/mL resin

Protein A Plus: >35mg human IgG/mL resin;

16-17mg mouse IgG/mL resin

Protein A Trisacryl Resin: >15mg human IgG/mL resin

Protein A UltraLink Resin: >16mg of human IgG/mL resin

Protein A Plus UltraLink Resin: >30mg human IgG/mL resin

Protein A MagnaBind Beads: >0.2mg rabbit IgG/mL beads

Protein A Coated Plates: ~4pmol rabbit IgG/well

Recombinant Protein A: ≥12mg human IgG/mL resin using the

IgG Buffer System

Ordering Information

Product #

Description

Pkg. Size

U.S.

Price

20333 Protein A Agarose

5mL

$ 337

20334 Protein A Agarose

25mL

$1050

20356 Protein A Columns

5 x 1mL

$ 385

44667 Protein A IgG Puriﬁcation Kit

Kit

$ 515

20338 Protein A Trisacryl Resin

5mL

$ 350

53139 Protein A UltraLink Resin

5mL

$ 340

22810 Protein A Plus Agarose

1mL

$ 120

22811 Protein A Plus Agarose

5mL

$ 400

22812 Protein A Plus Agarose

25mL

$1350

89924 Chromatography Cartridges, Protein A

2 x 1mL

$ 170

89925 Chromatography Cartridge, Protein A

1 x 5mL

$ 335

89952 NAb Protein A Plus Spin Columns

10 x 0.2mL

$ 222

89956 NAb Protein A Plus Spin Columns

5 x 1mL

$ 425

89960 NAb Protein A Plus Spin Column

1 x 5mL

$ 400

89948 NAb Protein A Plus Spin Puriﬁcation Kit

Kit

$ 270

89978 NAb Protein A Plus Spin Puriﬁcation Kit

Kit

$ 265

53142 Protein A UltraLink Plus Resin

5mL

$ 399

45202 Protein A Spin Plate

1 plate

$ 188

21348 MagnaBind Protein A Beads

5mL

$ 325

15130 Protein A, Clear 96-Well Plates

5 plates

$ 160

15132 Protein A, Clear 8-Well Strip Plates

5 plates

$ 178

15154 Protein A, White 96-Well Plates

5 plates

$ 178

15155 Protein A, Black 96-Well Plates

5 plates

$ 178

20365 Recombinant Protein A Agarose

5mL

$ 275

20366 Recombinant Protein A Agarose

25mL

$1001

Protein G

Protein G Beads – Quick Reference

Protein G Recombinant protein expressed in E. coli

(21.6kDa; two IgG binding sites)

Speciﬁcity

(Table 3)

Best for IgG from mouse, human, cow, goat and sheep;

poor for guinea pig, pig, dog and cat

Supports

Offered

Crosslinked 6% beaded agarose

UltraLink Biosupport

Magnetic particles (1-4μm)

Package

Formats

Resin slurries (3 sizes)

Chromatography Cartridges (2 sizes)

Packed Spin Columns (3 sizes)

Nab IgG Puriﬁcation Kits (2 sizes)

Storage 4°C, do not freeze

Capacity Protein G: 11-15mg human IgG/mL resin

Protein G UltraLink Resin: >20mg of human IgG/mL resin

Protein G Plus: >20mg human IgG/mL resin

Protein G UltraLink Resin: >20mg of human IgG/mL resin

Protein G Plus UltraLink Resin: >25mg human IgG/mL resin

MagnaBind Protein G Beads: >0.2mg rabbit IgG/mL beads

Protein G Coated Plates: ~2pmol rabbit IgG/well

Protein G Plus: >20mg human IgG/mL resin

Protein G Plus UltraLink Resin: >25mg human IgG/mL resin

Ordering Information

Product #

Description

Pkg. Size

U.S.

Price

20398 Protein G Agarose

2mL

$ 212

20399 Protein G Agarose

10mL

$ 760

20397 Protein G Agarose

25mL

$1654

89926 Pierce Chromatography Cartridges, Protein G

2 x 1mL

$ 190

89927 Pierce Chromatography Cartridge, Protein G

1 x 5mL

$ 390

89953 NAb Protein G Spin Columns

10 x 0.2mL

$ 251

89957 NAb Protein G Spin Columns

5 x 1mL

$ 455

89961 NAb Protein G Spin Columns

1 x 5mL

$ 425

89949 NAb Protein G Spin Puriﬁcation Kit

Kit

$ 310

89979 NAb Protein G Spin Puriﬁcation Kit

Kit

$ 368

21193 Pierce Recombinant Protein G

5mg

$ 300

53125 Protein G UltraLink Resin

2mL

$ 270

53126 Protein G UltraLink Resin

10mL

$ 910

53127 Protein G UltraLink Columns

2 x 2mL

$ 540

45204 Protein G Spin Plate

1 plate

$ 209

22851 Protein G Plus Agarose

2mL

$ 260

22852 Protein G Plus Agarose

10mL

$ 895

53128 Protein G Plus UltraLink Resin

2mL

$ 335

21349 MagnaBind Protein G Beads

5mL

$ 325

15131 Protein G, Clear 96-Well Plates

5 plates

$ 178

15133 Protein G, Clear 8-Well Strip Plates

5 plates

$ 195

15156 Protein G, White 96-Well Plates

5 plates

$ 195

15157 Protein G, Black 96-Well Plates

5 plates

$ 195

Antibody Puriﬁcation

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch ofﬁce or distributor.

Protein A/G

Protein A/G Beads – Quick Reference

Protein A/G Recombinant protein expressed in E. coli

(50.5kDa; six IgG-binding sites)

Speciﬁcity

(Table 3)

Best for polyclonal IgG from many species; poor for individual

subclasses that have highest afﬁnity for Protein A or Protein G.

Supports

Offered

Crosslinked 6% beaded agarose

UltraLink Biosupport

Package

Formats

Resin slurries (3 sizes)

Chromatography Cartridges (2 sizes)

Packed Spin Columns (3 sizes)

NAb IgG Puriﬁcation Kits (2 sizes)

Storage 4°C, do not freeze

Capacity Protein A/G: >7mg human IgG/mL resin

Protein A/G UltraLink Resin: >20mg human IgG/mL resin

Protein A/G Plus: >50mg human IgG/mL resin

Protein A/G Plus on UltraLink Support: >28mg human IgG/mL resin

Protein A/G Coated Plates: ~5pmol rabbit IgG/well

Ordering Information

Product #

Description

Pkg. Size

U.S.

Price

15138 Protein A/G, Clear 96-Well Plates

5 plates

$ 196

20421 Protein A/G Agarose

3mL

$ 315

20422 Protein A/G Agarose

15mL

$1150

89930 Pierce Chromatography Cartridges, Protein A/G

2 x 1mL

$ 299

89931 Pierce Chromatography Cartridge, Protein A/G

1 x 5mL

$ 399

89954 NAb Protein A/G Spin Columns

10 x 0.2mL

$ 260

89958 NAb Protein A/G Spin Columns

5 x 1mL

$ 460

89962 NAb Protein A/G Spin Column

1 x 5mL

$ 435

89950 NAb Protein A/G Spin Puriﬁcation Kit

Kit

$ 336

89980 NAb Protein A/G Spin Puriﬁcation Kit

Kit

$ 375

53132 Protein A/G UltraLink Resin

2mL

$ 270

53133 Protein A/G UltraLink Resin

10mL

$ 910

21186 Pierce Recombinant Protein A/G

5mg

$ 303

20423 Protein A/G Plus Agarose

2mL

$ 340

53135 Protein A/G Plus on UltraLink Support

2mL

$ 367

Protein L

Protein L Beads – Quick Reference

Protein L Recombinant protein expressed in E. coli (35.8kDa;

four IgG-binding sites)

Speciﬁcity

(Table 3)

Best for human or mouse monoclonal antibodies known to

have appropriate kappa light chains; poor for general-purpose

(polyclonal) IgG puriﬁcation

Supports

Offered

Crosslinked 6% beaded agarose

Package

Formats

Resin slurries (2 sizes)

Chromatography Cartridges (2 sizes)

Packed Spin Columns (3 sizes)

NAb IgG Puriﬁcation Kits (2 sizes)

Storage 4°C, do not freeze

Capacity Protein L: 5-10mg human IgG/mL resin

Protein L Plus: >10-20mg human IgG/mL resin

Ordering Information

Product #

Description

Pkg. Size

U.S.

Price

20510 Protein L Agarose

2mL

$248

20512 Protein L Agarose

10mL

$950

89928 Pierce Chromatography Cartridges, Protein L

2 x 1mL

$190

89929 Pierce Chromatography Cartridge, Protein L

1 x 5mL

$375

89955 NAb Protein L Spin Columns

10 x 0.2mL

$270

89959 NAb Protein L Spin Columns

5 x 1mL

$489

89963 NAb Protein L Spin Column

1 x 5mL

$459

89951 NAb Protein L Spin Puriﬁcation Kit

Kit

$365

89981 NAb Protein L Spin Puriﬁcation Kit

Kit

$410

21189 Pierce Recombinant Protein L

1mg

$121

20520 Protein L Plus Agarose

2mL

$365

15190 Protein L, Clear 96-Well Plates

5 plates

$191

For complete product details,

visit www.thermoscientiﬁc.com/pierce

or request the Antibody Puriﬁcation

Handbook (1601974)

Version 2

Thermo Scientiﬁc Pierce

Antibody Production and

Puriﬁcation Technical Handbook

For more information, or to download product instructions, visit www.thermoscientiﬁc.com/pierce

IgG Binding and Elution Buffers for

Protein A, G, A/G and L

Binding and Elution Steps in Afﬁnity Puriﬁcation

Afﬁnity puriﬁcation procedures involving interaction of an antibody

with its antigen generally use binding buffers at physiologic pH

and ionic strength. However, many antibody puriﬁcation methods

do not use the antibody-antigen interaction; rather, they involve

binding of antibodies by immobilized ligands that are not the

antigen. In such cases, optimal binding conditions are determined

by the unique properties of the antibody-ligand interaction, which

may be different from physiologic pH and ionic strength.

Once the binding interaction occurs (i.e., the antibody is “captured”

by the immobilized ligand), the support is washed with additional

buffer to remove nonbound components of the sample. Finally,