FPLC purification of his-tagged proteins—TALON Superflow Metal Affinity Resin
TALON his-tag purification resin lets you prepare exceptionally pure his-tagged proteins from bacterial, mammalian, yeast, and baculovirus-infected cells, under native or denaturing conditions. TALON is an immobilized metal affinity chromatography (IMAC) resin charged with cobalt, which binds to his-tagged proteins with higher specificity than nickel-charged resins. As a result, TALON resin delivers his-tagged proteins of the highest purity. In addition, each cobalt ion is bound to the resin at four sites, resulting in low metal-ion leakage.
Overview
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. 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 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—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.
Features
- Exhibits high affinity for his-tagged proteins
- Ideal for FPLC applications
- No copurification of proteins
- Resists metal leakage
- Performs well under a wide range of purification conditions
Applications
Purified recombinant his-tagged proteins can be used for:
- Crystallography
- Functional assays
- Structural investigations
- Other applications
