Scientific Background | Hexahistidine is the most widely used affinity tag in recombinant protein work. The supplied sequence is six consecutive histidine residues, and the tag functions by coordinating immobilised divalent metal ions, most commonly nickel or cobalt, through the imidazole nitrogens of those side chains. The free peptide is not used as a tag but as a reagent that acts on the same chemistry. Its principal application is as a competing ligand: like imidazole, it displaces His-tagged protein from a metal affinity column, and because it presents the same hexahistidine geometry as the tag itself it competes specifically rather than by sheer molar excess. It is also used to strip or regenerate resin, to quantify binding capacity, and as a blocking reagent to test whether an interaction attributed to a tagged protein is in fact mediated by the tag, which is a recognised artefact given that polyhistidine can bind metals and some surfaces on its own. It serves equally as a defined standard for anti-His antibodies in western blotting and ELISA, where a peptide of known composition is more convenient than a tagged protein whose concentration must be inferred. Note that because histidine protonates in the physiological pH range, binding behaviour is pH sensitive, which is worth controlling deliberately in any quantitative competition experiment. |
