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Protein Expression & Purification


Products >  Protein_Expression_and_Purification >  His-Tagged_Protein_Purification >  Cobalt_Column-Bacterial_Culture

His-Tagged Protein Purification from Bacterial Cultures—TALON Single Step

TALON Single Step Columns simplify purification of his-tagged proteins. These columns combine our exclusive TALON xTractor Buffer with the patented TALON resin, to consolidate the preliminary purification steps: cell lysis, centrifugation, and resin binding. These columns provide a convenient method of purifying material for preliminary characterization of your protein of interest.

Easy, Fast, and Pure

The entire process, from culture to purified protein, can be completed in less than 1 hour.

  1. Add the culture containing your his-tagged protein to the column and then mix for 20 minutes. The column contains a dried version of TALON xTractor Buffer, which lyses cells and releases your his-tagged target protein. Our proprietary TALON Resin binds his-tags with higher specificity than any other IMAC resin, so you are assured of obtaining a high degree of purification.
  2. After the unbound cellular debris is washed from the column, your highly pure his-tagged fusion protein is eluted using standard conditions.

Looking to purify smaller amounts of protein? The 5-ml columns routinely provide up to 0.5 mg of purified protein and the 20-ml columns regularly provide up to 3.0 mg of purified protein.

Value and Versatility

These time-saving columns are more convenient than the typical method of extracting and purifying his-tagged protein over a standard TALON Resin Column. Any 6xHis-, 6xHN-, or HAT-tagged protein can be purified using either a gravity flow or a spin column method. It is easy to run several columns in parallel at room temperature, to isolate various proteins at the same time.

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

Features

Simple purification of his-tagged proteins

  • Complete extraction and purification in under 1 hour
  • Purify target protein directly from bacterial culture
  • Ready-to-use, prepacked columns
  • Two sizes of columns available, for large and small scale purification

Applications

  • Preliminary protein characterization
  • Fast capture of his-tagged proteins

Reagents Compatible with TALON Resin

Reagent Acceptable Concentration
Beta-mercaptoethanola 10 mM (with caution)
CHAPSb 1% (with caution)
Ethanolc 30%
Ethylene glycol 30%
HEPES 50 mM
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.

Additional Information

Please see the product's Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Kit Components List to determine kit components. Certificates of Analysis and Kit Components Lists are located under the Documents tab.


 
 
Products
Cat. # Product Contents Size Price License Units Select
635632 TALON® Single Step Columns (20 ml) 10 Columns $302.00 License Statements
635628 TALON® Single Step Columns (5 ml) 25 Columns $286.00 License Statements
635631 TALON® Single Step Columns (5 ml) 2 Columns $50.00 License Statements
 

 

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