Polyhistidine-tags are often used for affinity purification of polyhistidine-tagged recombinant proteins expressed in
Escherichia coli [2] and other prokaryotic expression systems. Bacterial cells are harvested via
centrifugation and the resulting cell pellet lysed either by physical means or by means of detergents and
enzymes such as
lysozyme or any combination of these. At this stage raw lysate contains the recombinant protein among many other proteins originating from the bacterial host. This mixture is incubated with an affinity resin containing bound
divalent nickel or
cobalt ions, which are available commercially in different varieties. Nickel and cobalt have similar properties and as they are adjacent period 4 transition metals ((v.
iron triad)). These resins are generally sepharose/agarose functionalised with a
chelator, such as
iminodiacetic acid (Ni-IDA) and
nitrilotriacetic acid (Ni-NTA) for nickel and carboxylmethylaspartate (Co-CMA) for cobalt, which the polyhistidine-tag binds with
micromolar affinity. The resin is then washed with phosphate buffer to remove proteins that do not specifically interact with the cobalt or nickel ion. With Ni-based methods, washing efficiency can be improved by the addition of 20 mM
imidazole (proteins are usually eluted with 150-300 mM imidazole). Generally nickel-based resins have higher binding capacity, while cobalt-based resins offer the highest purity. The purity and amount of protein can be assessed by
SDS-PAGE and
Western blotting.
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