- Why tag a protein?
- Tech note: cobalt resin
- Purification methods overview
- Simplified purification of active, secreted his-tagged proteins
- Overview: His60
- Tech note: Capturem technology
- Tech note: Capturem large volume
- Magnetic beads
- TALON resin selection guide
- Selection guide: His60 resin
- FAQs: TALON
- Video: Capturem his maxiprep
- Video: Capturem his miniprep
- Visual protocol: Capturem his maxiprep
- Visual protocol: Capturem his miniprep
- Capturem nickel column reagent compatibility
- TALON reagent compatibility
- His60 reagent compatibility
- TALON: Native vs denaturing purification
- Protocol: denaturing purification with TALON resin, imidazole elution
- Protocol: native purification with TALON resin, imidazole elution
- Protocol: native purification with TALON resin, pH elution
Choice of native or denaturing purification conditions and use of reducing agents with TALON resin
Native or denaturing?—Selecting the right purification conditions
Deciding whether to use native or denaturing purification conditions depends on protein localization, solubility, accessibility of the histidine tag, downstream applications, and whether preservation of biological activity is required. 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.
Purifying a protein under native conditions (see example below) is the most efficient way to preserve its biological activity, but requires that the protein is 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
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 below)—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 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, 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 in the presence of β-mercaptoethanol.
TALON resin application protocols
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