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Protein Expression & Purification

Products >  Protein_Expression_and_Purification >  His-Tagged_Protein_Purification >  Cobalt_Columns-Gravity

HisTALON Gravity Columns—Fast, Easy His-Tagged Protein Purification

TALON Metal Affinity Resin is a durable immobilized metal affinity chromatography (IMAC) resin that has a remarkable affinity and specificity for his-tagged proteins. TALON Resin is compatible with all commonly used IMAC reagents and allows protein purification under native or denaturing conditions.

TALON Metal Affinity Resin uses cobalt ions for purifying recombinant his-tagged proteins. TALON is a tetradentate chelator charged with cobalt, and is specific for his-tagged proteins. The TALON reactive core, containing cobalt, has strict requirements for the spatial positioning of histidines. Only adjacent histidines or specially positioned, neighboring histidines are able to bind the cobalt in this reactive core. In nickel-based resins (i.e., Ni-NTA Resin), these spatial requirements are less strict. Therefore, nickel-based resins are also able to bind histidines located in places other than the protein’s his-tag.

Clontech offers ready-to-use HisTALON Gravity Columns for the efficient purification of his-tagged proteins from bacterial, mammalian, and baculovirus-infected cells, using gravity-flow-based protocols. The columns are prepacked with our TALON Resin and can absorb more than 20 mg of his-tagged AcGFP1. These columns enable fast, easy, and reproducible chromatographic separation—and can be regenerated for multiple uses. However, we recommend that you reuse a column only to purify different batches of the same protein. If you plan to purify multiple proteins using the same column, you must utilize the “Complete Regeneration” method described in the User Manual.

Choice of Native or Denaturing Purification Conditions

TALON Resin retains its protein binding specificity and yield under a variety of purification conditions. It is stable under both denaturing and native (nondenaturing) conditions (see flow chart). Deciding whether to use native or denaturing purification conditions depends on protein location, solubility, accessibility of the his tag, downstream applications, and preservation of biological activity.

  • Native Conditions

    Purifying a protein under native conditions (see example) is the most efficient way to preserve its biological activity, but requires that the protein be soluble. Advantages include:

    • Eliminating the renaturation step at the end of the purification, saving time, and preventing significant loss of activity
    • Retaining the ability to copurify enzyme subunits, cofactors, and associated proteins
  • Denaturing Conditions

    Because proteins that are overexpressed in prokaryotic systems sometimes form insoluble aggregates called inclusion bodies, you may need to purify proteins under denaturing conditions (see example)—using strong denaturants such as 6 M guanidinium or 8 M urea to enhance protein solubility. Advantages include:

    • Complete solubilization of inclusion bodies and his-tagged proteins
    • Improved binding to the matrix and reduced nonspecific binding, due to full exposure of the his tag

His-tagged proteins purified under denaturing conditions can be used directly in subsequent applications, or may need to be renatured and refolded. Protein renaturation and refolding can be performed prior to elution from the column. However, yields of recombinant proteins will be lower than under native conditions, because urea and guanidinium molecules compete with histidines for binding to metal.

Use of Reducing Agents

Purification with TALON Resin may be carried out in the presence of β-mercaptoethanol (see example), but not DTT or DTE, to preserve reduced sulfhydryl (-SH) groups that are important for the biological activity and structure of a given protein. TALON provides higher yields than Ni-NTA (see example) in the presence of β-mercaptoethanol.

  At-A-Glance   Documents   Images & Data   Resources


  • Easy-to-use prepacked gravity columns
  • Maximize yield of biologically active protein
  • Superior purity & high yields with TALON Resin


Purified recombinant his-tagged proteins can be used for:

  • Crystallography
  • Functional assays
  • Structural investigations
  • Other applications

Reagents Compatible with TALON Resin

Reagent Acceptable Concentration
Beta-mercaptoethanola 10 mM (with caution)
CHAPSb 1% (with caution)
Ethanolc 30%
Ethylene glycol 30%
Glycerol 20%
Guanidine hydrochloridea 6 mM
Imidazoled 200 mM at pH 7.0–8.0, for elution
KCl 500 mM
MES 20 mM
MOPS 50 mM
NaCl 1.0 M
NP-40 1%
SDSb 1% with caution
Trise 50 mM
Triton-X 100 <1%
Urea 8 M

a 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.
b 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, even at low concentrations.
c Ethanol may precipitate proteins, causing low yields and column clogging.
d 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.
e Tris coordinates weakly with metal ions, causing a decrease in capacity.

Reagents Incompatible with TALON Resin

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), EGTA (ethylene glycol tetraacetic acid), and PEI
    NOTE: Although you can use EDTA at indicated points, it must be removed from the sample by gel filtration prior to applying the sample to TALON Resins.

Cat. # Product Contents Size Price License Units Select
635654 HisTALON™ Gravity Column Purification Kit 5 x 1 mL Columns $319.00 License Statements
635655 HisTALON™ Gravity Columns 5 x 1 mL Columns $120.00 License Statements


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635651 HisTALON™ Buffer Set 20 Purifications $225.00
635673 ProteoGuard™ EDTA-Free Protease Inhibitor Cocktail 10 x 100 uL $140.00

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