TALON® Metal Affinity Resins User Manual

Clontech Laboratories, Inc.

A Takara Bio Company

1290 Terra Bella Avenue, Mountain View, CA 94043, USA

U.S. Technical Support: tech@clontech.com

United States/Canada

800.662.2566

Asia Pacific

+1.650.919.7300

Europe

+33.(0)1.3904.6880

Japan

+81.(0)77.543.6116

Clontech Laboratories, Inc.

TALON

Metal Affinity

Resins User Manual

Cat. Nos. Many

PT1320-1 (031716)

TALON

Metal Affinity Resins User Manual

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031716 Clontech Laboratories, Inc. A Takara Bio Company Page 2 of 15

Table of Contents

I. Introduction ..................................................................................................................................................................... 2

II. List of Components ......................................................................................................................................................... 4

III. Additional Materials Needed .......................................................................................................................................... 5

IV. Sample Preparation Protocols ......................................................................................................................................... 6

A. Protocol: Sample Preparation to Isolate Native Proteins ......................................................................................... 6

B. Protocol: Sample Preparation to Isolate Denatured Proteins ................................................................................... 7

V. Protein Purification Protocols ......................................................................................................................................... 7

A. Protocol: Batch/Gravity-Flow Column Purification ................................................................................................ 7

B. Protocol: Large-Scale Batch Purification ................................................................................................................. 9

VI. TALON Resin Regeneration and Storage ..................................................................................................................... 10

A. Important Precautions ............................................................................................................................................ 10

B. Protocol: Resin Regeneration & Storage ................................................................................................................ 10

VII. Troubleshooting ............................................................................................................................................................ 10

Appendix A: Reagent Compatibilities and Incompatibilities ............................................................................................... 14

VIII. References ..................................................................................................................................................................... 15

Table of Tables

Table 1. Physiochemical Properties of TALON Resins .......................................................................................................... 3

Table 2. Reagent Compatibility ............................................................................................................................................ 14

I. Introduction

A. Summary

TALON Metal Affinity Resin is a durable immobilized metal affinity chromatography (IMAC) resin, consisting

of a tetradentate chelator charged with cobalt, which has a remarkable affinity and specificity for his-tagged

proteins (Chaga et al., 1994; Froelich et al., 1996; Hochuli et al., 1987 & 1988; Porath et al., 1975; Stephens et

al., 1997). The stable chelation of the Co

ion, combined with the specificity of the TALON reactive core, deliver

unmatched purity. This resin is compatible with all commonly used IMAC reagents and allows protein

purification under native or denaturing conditions, and in the presence of ß-mercaptoethanol. It is available as a

50% slurry in three different formats.

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B. Overview of TALON Resin Formats

 TALON Metal Affinity Resin is useful for batch and low-pressure chromatographic applications.

This resin utilizes Sepharose CL-6B (GE Healthcare), a durable substrate that performs very well

under native and denaturing conditions in centrifuge-mediated purification schemes. The large pore

size resin has a high-binding capacity.

 TALON Superflow Resin is specifically designed for quick, effective purification of his-tagged

proteins at high flow rates (up to 5 ml per cm

per min) and medium pressure (up to 150 psi). This

resin is recommended if short purification times are essential, or if purification protocols developed at

bench scale will be scaled up for larger volumes. It utilizes Superflow-6 (Sterogene Bioseparations,

Inc.), an agarose-based medium featuring a unique polysaccharide composition that resists biological

degradation. Superflow-6 beads are also stabilized by a chemical cross-linking reaction that allows

flow rates up to 10 times higher than are possible with regular cross-linked beads.

 TALON CellThru Resin is a novel IMAC resin for purifying his-tagged proteins from crude cell

lysates, sonicates, and fermentation liquids. The larger bead size of TALON CellThru Resin (300–

500 µm) permits cellular debris to flow through the column, eliminating the need for high-speed

centrifugation. Destabilizing factors are removed more quickly with this resin than with other IMAC

resins, because the number of steps is reduced.

Table 1. Physiochemical Properties of TALON Resins

Features

TALON Resin

TALON Superflow

Resin

TALON CellThru Resin

Batch/gravity flow

applications

Yes

FPLC applications

Yes

Scale

Analytical, preparative,

production

Analytical, preparative,

production

Preparative, production

Capacity*

(mg protein/ ml adsorbent)

5–15

5–18

5–10

Matrix

Sepharose 6B-CL

(6% crosslinked

agarose)

Superflow

(6% crosslinked

agarose)

Uniflow

(4% crosslinked

agarose)

Bead size (µm)

45–165

60–160

300–500

Maximum linear flow rate

(cm/hr)**

3,000

800

Maximum volumetric flow

rate (ml/min)**

0.5

Recommended volumetric

flow rate (ml/min)

0.3

1.0–5.0

Maximum pressure

2.8 psi

0.2 bar

0.02 MPa

150 psi

10 bar

0.97 MPa

9 psi

0.62 bar

0.02 MPs

pH stability (duration)

2–14 (2 hr)

3–14 (24 hr)

2–14 (2 hr)

3–14 (24 hr)

2–14 (2 hr)

3–14 (24 hr)

Protein exclusion limit (Da)

4 x 10

2 x 10

* The binding capacity of individual proteins may vary.

** For washing and elution only.

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II. List of Components

TALON Resin, TALON Superflow Resin, and TALON CellThru Resin are supplied as 50% (w/v) slurries in nonbuffered

20% ethanol. Please note that during shipping and storage, the resin will settle; thus, we recommend that you thoroughly

resuspend it before aliquotting. 2 ml of homogeneously resuspended slurry will provide 1 ml of resin with a binding

capacity of at least 5 mg of his-tagged protein.

Store all of these resins, columns and buffers at 4°C unless otherwise indicated. Do not freeze TALON Resins.

A. Resins

TALON Metal Affinity Resin

Cat. No.

Amount

635501

10 ml

635502

25 ml

635503

100 ml

635504

250 ml

TALON Superflow Resin

Cat. No.

Amount

635506

25 ml

635507

100 ml

TALON CellThru Resin

Cat. No.

Amount

635509

10 ml

635510

100 ml

B. Columns

TALON 2 ml Disposable Gravity Columns*

Cat. No.

Amount

635606

50 x 2 ml columns

TALON CellThru Disposable Columns*

Cat. No.

Amount

635513

20 x 10 ml columns

*Use one of these empty disposable columns for Step 3.f of the Batch/Gravity-Flow Column Purification Protocol (Section V.A).

C. Buffers

xTractor™ Buffer*

Cat. No.

Amount

635656

100 ml

635671

250 ml

635625

2 x 250 ml

xTractor Buffer Kit (Cat. No. 635623)

200 ml

1X xTractor Buffer*

2.5 ml

100X Lysozyme

400 µl

DNase (5 units/ml)**

HisTALON™ Buffer Set (Cat. No. 635651)

2 x 250 ml

HisTALON Equilibration Buffer

200 ml

HisTALON Elution Buffer

100 ml

HisTALON xTractor Buffer*

* xTractor Buffer is equivalent to HisTALON xTractor Buffer.

**Store DNase I at –20°C.

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III. Additional Materials Needed

A. Buffer Preparation

Use the buffers supplied in the HisTALON Buffer Set (Section II.B) to extract and purify proteins under native

and denaturing conditions as follows:

1. Native Conditions

 Lysis Buffer: use HisTALON xTractor Buffer (or TALON xTractor Buffer)

 Equilibration Buffer: use HisTALON Equilibration Buffer

 Wash Buffer: mix 660 µl of HisTALON Elution Buffer with 9.34 ml of HisTALON

Equilibration Buffer

 Elution Buffer: use HisTALON Elution Buffer

2. Denaturing Conditions

 Equilibration Buffer: Add guanidine-HCl to HisTALON Equilibration Buffer to a final

concentration of 6 M, and, if necessary, readjust the pH to 7.4.

 Wash Buffer: Add guanidine-HCl to Wash Buffer (III.A) to a final concentration of 6 M,

and, if necessary, readjust the pH to 7.4.

 Elution Buffer: Add guanidine-HCl to HisTALON Elution Buffer to a final concentration of

5.4 M, and, if necessary, adjust the pH to 7.4.

3. Resin Regeneration

 Regeneration Buffer (pH 5.0): 20 mM MES (2-(N-morpholine)-ethanesulfonic acid), 0.3 M

sodium chloride

4. Protease Inhibitors

ProteoGuard™ EDTA-Free Protease Inhibitor Cocktail (Cat. No. 635673)

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IV. Sample Preparation Protocols

Please read each protocol completely before starting. Successful results depend on understanding and performing

the following steps correctly.

IMPORTANT: We strongly recommend using ProteoGuard™ EDTA-Free Protease Inhibitor Cocktail (Cat.

No. 635673) when preparing your protein extract. Add 10 μl of Protease Inhibitor Cocktail per ml of lysis buffer

[xTractor Buffer for native proteins (Section IV.A) or denaturing Equilibration Buffer for denatured proteins

(Section IV.B)] before lysing cells to yield a 1X final concentration of inhibitors. For more information, refer to

the ProteoGuard Protocol (type PT5140-2 in the keyword field at www.clontech.com/manuals).

A. Protocol: Sample Preparation to Isolate Native Proteins

This procedure has been optimized for extraction of native proteins from fresh or frozen bacterial cell pellet. The

volumes of this extraction can be adjusted, as long as 20 ml of xTractor Buffer are used per 1 g of cell pellet.

1. Add 20 ml of xTractor Buffer to 1 g of bacterial cell pellet. Gently pipet up and down to fully

resuspend the pellet.

NOTE: A log-phase culture of E. coli (O.D.=0.6–0.8) when induced for 2–4 hours, would be

expected to provide ~20–40 mg bacterial pellet from 2 ml of the culture.

2. [Optional]: Add 40 µl of 5 units/µl DNase I solution and 200 µl of 100X lysozyme solution.

NOTES:

 DNase I reduces lysate viscosity, allowing more efficient cellular debris removal. DNase can be

used without lysozyme—but cells treated with lysozyme must also be treated with DNase I.

 Lysozyme helps to fully disrupt bacterial walls, and thus to extract high molecular weight

proteins (>40 KDa). However, lysozyme should be omitted for mammalian extraction procedures

as well as when lysozyme interferes with your protein's functionality.

 If the lysozyme solution forms a precipitate, resuspend the contents of the bottle and apply 200 μl

of suspension directly to the mix—or (optionally) centrifuge 200 μl of lysozyme solution for 5

min at 14,000 rpm, and use the supernatant for the lysis.

3. Mix gently, pipetting up and down several times.

4. Incubate with gentle shaking for 10 min at room temperature or at 4°C.

NOTE: At the end of this incubation period, there should be no visible particles. If cell pellet

fragments are present, resuspend them by pipetting solution up and down and incubating for an

additional 1–2 min.

5. The resulting cell lysate can now be applied directly to a TALON CellThru Column, or the lysate

supernatant can be applied to any other TALON Resin column after centrifuging (10,000–12,000 x g

for 20 min at 4°C).

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B. Protocol: Sample Preparation to Isolate Denatured Proteins

1. Harvest 20–25 ml of cell culture by centrifugation at 1,000–3,000 x g for 15 min at 4°C.

2. Resuspend pellet in 2 ml of denaturing Equilibration Buffer (Section III.A.2) per 20–25 ml of

culture.

3. Gently agitate or stir the sample until it becomes translucent.

4. Centrifuge the sample at 10,000–12,000 x g for 20 min at 4°C to pellet any insoluble material.

5. Carefully transfer the supernatant (the clarified sample) to a clean tube without disturbing the pellet.

6. Set aside a small portion of the clarified sample for SDS/PAGE analysis before starting purification.

NOTE: Samples containing 6 M guanidinium must be dialyzed overnight against buffer containing

8 M urea before loading on a gel.

V. Protein Purification Protocols

Please read each protocol completely before starting. Successful results depend on understanding and performing the

following steps correctly. Use the appropriate set of buffers depending on whether you are purifying protein under native

or denaturing conditions (Section III.A).

A. Protocol: Batch/Gravity-Flow Column Purification

For IMAC columns using TALON, we recommend a hybrid batch/gravity-flow procedure. This method combines

the speed and convenience of a batch procedure with the higher purity of the gravity-flow column method. In this

hybrid procedure, the binding and initial washing steps are performed in a batch format to save time, eliminate

extraneous debris, and avoid column clogging. After the initial washes, the resin is transferred to a column for

additional washing and protein elution.

1. Resin Equilibration

a. Thoroughly resuspend the TALON Resin.

b. Immediately transfer the required amount of resin suspension to a sterile tube that will

accommodate 10–20 times the resin bed volume.

c. Centrifuge at 700 x g for 2 min to pellet the resin.

d. Remove and discard the supernatant.

e. Add 10 bed volumes of Equilibration Buffer and mix briefly to pre-equilibrate the resin.

f. Recentrifuge at 700 x g for 2 min to pellet the resin. Discard the supernatant.

g. Repeat Steps e and f.

2. Sample Application

a. Add crude lysate (TALON CellThru) or clarified sample (TALON & TALON Superflow)

from Section IV to the resin.

b. Gently agitate at room temperature or on ice for 20 min on a platform shaker to allow the his-

tagged protein to bind the resin.

NOTE: Incubation on ice will decrease proteolysis.

c. Centrifuge at 700 x g for 5 min. Carefully remove as much supernatant as possible without

disturbing the resin pellet.

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3. Washing

a. Wash the resin by adding 10–20 bed volumes of Equilibration Buffer. Gently agitate the

suspension at room temperature or on ice for 10 min on a platform shaker for thorough

washing.

b. Centrifuge at 700 x g for 5 min.

c. Remove and discard the supernatant.

d. Repeat Steps a–c with 10–20 bed volumes of Equilibration Buffer

e. Add one bed volume of Equilibration Buffer to the resin, and resuspend by vortexing.

NOTE: Steps f–h can be performed on ice or at room temperature, but incubation on ice will

decrease proteolysis.

f. Transfer the resin to a 2 ml gravity-flow column with an end-cap in place, and allow the resin

to settle out of suspension.

g. Remove the end-cap and allow the buffer to drain until it reaches the top of the resin bed,

making sure no air bubbles are trapped in the resin bed.

h. Wash column once with 5 bed volumes of Wash Buffer.

4. Elution & Analysis

a. Elute the his-tagged protein by adding 5 bed volumes of Elution Buffer to the column.

Collect the eluate in 500 μl fractions.

NOTE: Under most conditions, the majority of the his-tagged protein will be recovered in the

first two bed volumes.

b. Use spectrophotometric and SDS-PAGE analysis to determine which fraction(s) contain(s)

the majority of the his-tagged protein.

NOTE: Use a Bradford protein assay (Bradford, 1976) or UV absorbance at 280 nm.

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B. Protocol: Large-Scale Batch Purification

This method purifies his-tagged proteins faster than gravity-flow columns; however, batch washes remove

impurities less efficiently than gravity-flow columns. Therefore, they require larger wash buffer volumes to obtain

pure his-tagged proteins.

1. Resin Equilibration

a. Thoroughly resuspend the TALON Resin.

b. Transfer the required amount of resin to a glass filter with a pore size of 10–20 μm.

c. Apply a vacuum to the filter to remove excess ethanol.

d. Add 5 bed volumes of deionized water to the resin and apply vacuum.

e. Add 5 bed volumes of Equilibration Buffer to the resin and apply vacuum.

f. Repeat Step e two times.

2. Sample Application

a. Add crude lysate (TALON CellThru) or clarified sample (TALON & TALON Superflow) to

the resin and mix for 3–5 min.

b. Apply vacuum and collect the filtrate.

3. Washing

a. Wash the resin by adding 10–20 bed volumes of Equilibration Buffer. Gently agitate the

suspension at room temperature for 10 min on a platform shaker to promote thorough

washing.

b. Apply vacuum to remove buffer.

c. Repeat the above wash (Steps a–b) 2–3 times with 10–20 bed volumes of Equilibration

Buffer.

d. [Optional]: If necessary, repeat Step c under more stringent conditions using 5 mM

imidazole in Equilibration Buffer.

4. Elution & Analysis

a. Elute the his-tagged protein by adding 5 bed volumes of Elution Buffer.

b. Gently agitate suspension at room temperature for 5 min.

c. Apply vacuum, and collect the purified his-tagged protein.

d. Repeat Steps a–c two times, collecting separate fractions.

e. Use spectrophotometric analysis (absorbance at 280 nm) or a Bradford protein assay and

SDS-PAGE analysis to determine which fraction(s) contain(s) the majority of the his-tagged

protein.

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VI. TALON Resin Regeneration and Storage

Generally, reuse TALON Resins 3–4 times before discarding. The exact number of uses varies among

preparations because of differences in redox potential, organic complexity, and debris content. To avoid possible

cross-contamination, use a particular aliquot of resin to purify a single type of his-tagged protein.

A. Important Precautions

 Do not store TALON Resin in denaturants such as 6 M guanidinium.

 Do not store TALON Resin with bound imidazole; the resin should be washed with 2-(N-

morpholine)-ethanesulfonic acid (MES) buffer (pH 5.0) before reuse to remove the bound imidazole.

B. Protocol: Resin Regeneration & Storage

1. Wash resin with five bed volumes of 20 mM MES buffer (pH 5.0) containing 0.3 M NaCl.

2. Rinse resin with five bed volumes of distilled H

3. Store resin at 4°C in 20% nonbuffered ethanol containing 0.1% sodium azide.

VII. Troubleshooting

A. Loading/Washing

Description of Problem

Possible Explanation

Solution

Protein elutes in the wash

buffer

Problems with vector construction

Ensure that the protein and tag are in-frame.

Buffer compositions are not

optimal

Check the pH and composition of all buffers. Use

a lower-stringency wash buffer for all washing

steps.

For example, slightly increase the pH of the wash

buffer.

Protein is degraded during

extraction

 Perform initial purification step more quickly.

 Use mild extraction conditions in the presence

of ProteoGuard EDTA-Free Protease Inhibitor

Cocktail (Cat. No. 635673).

 For expressed proteins, make a C-terminal

construct.

Reagent interferes with binding

 Check Appendix A: Reagent Compatibilities

 Dilute an aliquot of lysate (1:10), or sonicate,

and check binding on a small scale.

 Try using a different his-tagged protein as a

control.

Tag is not accessible under native

conditions

 If the “under native conditions” protein fails to

bind under native conditions, treat a small

aliquot (<1 ml) with 6 M guanidinium and bind

to 50 μl of TALON and then proceed with

protein purification.

 If the target protein is now bound to the resin,

then try to move the tag to the other terminus

of the protein where it may be more exposed

under native conditions.

 Add small amounts of nonionic detergent(s) to

improve tag accessibility.

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B. Elution

Description of Problem

Possible Explanation

Solution

High amount of coeluted

impurities

Insufficient wash

Use larger volumes of Wash Buffer

Buffer compositions are not

optimal

 Check buffers used for sample preparation

and wash steps.

 Check pH. The Wash Buffer should be pH 7.4.

Contaminants will coelute in buffers with pH

<7.0.

 Increase the volume of wash buffer and

continue to wash resin bed until the A

280

drops

to zero.

 Increase counterion concentration up to 0.5 M

NaCl or KCl to inhibit nonspecific ionic

interactions.

 Add small amounts of nonionic detergent(s);

this is particularly important when isolating

proteins from a eukaryotic expression system.

 Add ethylene glycol or glycerol to inhibit

nonspecific hydrophobic interactions.

Resin was used in large excess

Estimate the fusion protein content of your lysate

and use only as much resin as required. (See

Table III for binding capacities of TALON resins.)

Proteolytic product

Use mild extraction conditions in presence of

ProteoGuard EDTA-Free Protease Inhibitor

Cocktail (Cat. No. 635673).

Covalent attachment (Cys-Cys,

disulfide bonds) of impurities to

the protein

Use 5–10 mM of β-ME in the Wash Buffer

Copurifying histidine-rich proteins

 For HAT- or his-tagged proteins, use

enterokinase to remove the HAT tag and

rerun IMAC with the mixture. Target protein

will pass through the column, while impurities

and tag will be adsorbed. (Remove chelating

ligands by gel filtration before loading the

proteolytic mixture onto TALON Resin.)

 Buffer pH is not optimal. Adjust pH as needed.

 Use second purification scheme, such as size

exclusion, ion exchange, hydrophobic

chromatography, etc.

Protein sample is too

concentrated and/or viscous

 Dilute sample 1:5 or 1:10 with additional buffer

and centrifuge again before proceeding.

 Digest sample for 20–30 min at room

temperature with 2.5 μg/ml of DNase I, taking

into account that proteolytic activity is much

higher at room temperature.

 Alternatively, dilute the sample fivefold with

Equilibration Buffer before applying it to the

resin. This procedure should not significantly

affect recovery.

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B. Elution (continued)

Description of Problem

Possible Explanation

Solution

Column ceases to flow

Frit or filter is clogged with

subcellular debris

Change column filters and centrifuge sample at

12,000 x g for 20–30 min at 4°C

The lower resin bed support may

be clogged with cellular debris

 Remove resin from clogged column and

resuspend. Then wash it in a batch format

and transfer to a fresh column.

 Use a syringe filled with wash buffer or

reverse the pump on the column to gently

run the column backwards. In addition, test

for tubing blockages in a similar manner.

Apply gentle pressure. Do not exceed a

1 drop/sec flow rate.

His-tagged proteins do not elute

Elution Buffer is not optimal

 Elute with 150 mM imidazole or pH 4.0 buffer.

 For proteins that will not elute otherwise, you

can strip off the protein using 100 mM EDTA

(pH 8.0). (This will remove the Co

from the

resin and deposit it in your protein sample.)

 Add 1–5 mM β-ME to reduce disulfide

linkages. Supplement buffer with 1% nonionic

detergent.

 Purify his-tagged protein under denaturing

conditions.

C. Changes in Resin

Description of Problem

Possible Explanation

Solution

White or pale-colored resin

Presence of chelators in the

sample causing loss of Co

Remove chelators from sample by gel filtration

and use fresh resin.

Gray or brown resin

TALON Resin was overexposed to

reducing agents or high

concentration of β-ME

Completely remove reducing agents, such as

DTE or DTT, or by gel filtration chromatography

in the presence of β-ME. Reduce β-ME

concentration (≤5 mM).

Resin particles aggregate or

exhibit a change in consistency

DNA crosslinking

 Increase ionic strength of the buffers by

using ≤ 500 mM NaCl.

 Vigorously sonicate samples before loading

to shear DNA.

 Pretreat sample with 100 μg/ml DNase I at

30°C for 30 min.

 Dilute sample 1:5–1:10 with buffer before

loading on column.

 Avoid long-term storage of resin in

denaturants.

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D. Analysis

Description of Problem

Possible Explanation

Solution

High background on silver-

stained gels

Nucleic acid contaminant

 Supplement buffer with 0.5 M NaCl or KCl.

Repeat purification

 Shear DNA more vigorously.

 Use DNAse I in the extraction procedure.

Nonfunctional protein

Protein was damaged by

sonication.

 Conduct a time-course assay to determine

the minimum sonication time needed to

disrupt the cells while maintaining the

native protein/enzyme function.

For example, sonicate samples at a

medium-high setting for 0, 20, and 30 sec.

Then perform protein or enzyme functional

assays and measure the A

280

of each

sample.

 Perform the lysis or sonication procedure

on ice.

Protein has degraded

 Keep protein samples at 4°C during

purification

 Reduce purification time for initial steps.

 Add ProteoGuard EDTA-Free Protease

Inhibitor Cocktail (Cat. No. 635673).

Protein may not be folded properly

Try purifying protein under denaturing

conditions, then try refolding

(Lin et al., 2007)

Protein may require a

posttranslational modification to be

active

In this case, it may be necessary to change the

expression system (e.g., switch from E. coli to

insect or mammalian cells.)

E. Resin Reuse

Description of Problem

Possible Explanation

Solution

Binding drops below original

capacity

Lysate contains naturally occurring

reducing agent or a nonspecific

polyanion may be obscuring the

metal binding sites.

 Use a larger volume of the previously-used

resin.

 Replace used resin with fresh resin.

 Wash resin with 6 M guanidinium (pH 5.0)

and 1% Triton X-100 or SDS, and re-

equilibrate before use.

Resin is dirty or has not been fully

regenerated.

Resin has been damaged or has worn out.

TALON Resins can be reused at least 3–4

times with proper handling.

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Appendix A: Reagent Compatibilities and Incompatibilities

A. Compatible Reagents

Table 2 shows the maximum concentrations of each reagent tested at Clontech. Higher levels may be

acceptable, but they should be tested before use. Note that some of these reagents may partially or

completely denature your protein.

Table 2. Reagent Compatibility

Reagent

Acceptable Concentration

ß-Mercaptoethanol

CHAPS

Ethanol

Ethylene glycol

HEPES 50 mM

Glycerol

Guanidinium

Imidazole

KCl

MES

MOPS

NaCl

NP-40

SDS

TRIS

Triton-X 100

Urea

10 mM (with caution)

1% (with caution)

30%

50 mM

20%

6 M

200 mM at pH 7.0–8.0, for elution

500 mM

20 mM

50 mM

1.0 M

1% with caution

50 mM

<1%

8 M

Use resin immediately after equilibrating with buffers containing these reagents. Otherwise, the resin will change color.

Do not store resin in buffers containing these reagents.

Ionic detergents like CHAPS (3-[(3-Cholamidopropyl)-dimethylammonio]-1-propane-sulfonate), SDS (sodium dodecyl

sulfate), and sarkosyl are compatible up to 1%. However, due to their charged nature, you should anticipate

interference with binding.

Ethanol may precipitate proteins, causing low yields and column clogging.

Imidazole cannot be used at concentrations higher than 5–10 mM for loading his-tagged proteins, because it

competes with the histidine side chains (imidazole groups) for binding to the immobilized metal ions.

TRIS coordinates weakly with metal ions, causing a decrease in capacity.

B. Incompatible Reagents

These reagents are incompatible at any concentration:

 DTT (dithiothreitol) and DTE (dithioerythritol)

NOTE: Using strong reducing agents will interfere with cobalt metal ion binding to the resin.

 EDTA (ethylenediaminetetraacetic acid) and EGTA (ethylene glycol-bis([β-amino-ethyl ether])

NOTE: Although you can use EDTA at indicated points, it must be removed from the sample by gel

filtration prior to applying it to TALON Resins.

TALON

Metal Affinity Resins User Manual

PT1320-1 www.clontech.com

031716 Clontech Laboratories, Inc. A Takara Bio Company Page 15 of 15

VIII. References

Bradford, M. M. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing

the principle of protein-dye binding. Anal. Biochem. 72:248–254.

Chaga, G., Widersten, M., Andersson, L., Porath, J., Danielson, U. H. & Mannervik, B. (1994) Engineering of a metal

coordinating site into human glutathione transferase M1-1 based on immobilized metal ion affinity chromatography of

homologous rat enzymes. Protein Eng. 7(9):1115–1119.

Froelich, C. J., Turbov, J. & Hanna, W. (1996) Human perforin: rapid enrichment by immobilized metal affinity

chromatography (IMAC) for whole cell cytotoxicity assays. Biochem. Biophys. Res. Commun. 229(1):44–49.

Hochuli, E., Döbeli, H. & Schacher, A. (1987) New metal chelate adsorbent selective for proteins and peptides containing

neighboring histidine residues. J. Chromatogr. 411:177–184.

Hochuli, E., Bannwarth, W., Döbeli, H., Gentz, R. & Stüber, D. (1988) Genetic approach to facilitate purification of novel

recombinant proteins with a novel metal chelate adsorbent. Bio/Technology 6:1321–1325.

Porath, J., Carlsson, J., Olsson, I. & Belfrage, G. (1975) Metal chelate affinity chromatography, a new approach to protein

fractionation. Nature 258:598–599.

Stephens, L. R., Eguinoa, A., Erdjument-Bromage, H., Lui, M., Cooke, F., Coadwell, J., Smrcka, A. S., Thelen, M.,

Cadwallader, K., Tempst, P. & Hawkins, P. T. (1997) The G beta gamma sensitivity of a PI3K is dependent upon a tightly

associated adaptor, p101. Cell 89(1):105–114.

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