His-tag

N- or C-terminal His-tags may also be followed or preceded, respectively, by a suitable amino acid sequence that facilitates removal of the polyhistidine-tag using endopeptidases.

[3] Subsequent studies have revealed that among amino acids constituting proteins, histidine is strongly involved in the coordination complex with metal ions.

[10][11] Most commonly, a polyhistidine tag is fused at the N-terminus or C-terminus of a protein and is attached via a short flexible linker, which may contain a protease cleavage site.

[10][9] Less commonly, tags can be added at both the N- and C-termini or inserted at an intermediate part of a protein, such as within an exposed loop.

[13] Various carrier matrices bound to a solid resin support are on the market and these can be subsequently charged with a metal cation.

Divalent cation M2+ (M = Mn, Fe, Co, Ni, Cu, Zn etc) transition metal imidazole complexes are most frequently used for this purpose.

The compound added at high concentration replaces virtually all carrier-bound protein which is thus eluted from the carrier.

When the pH decreases, the histidine residue is protonated and can no longer coordinate the metal tag, allowing the protein to be eluted.

Protein purity can be improved by the addition of a low (20-40 mM) concentration of imidazole to the binding and/or wash buffers.

These impurities can be eliminated using additional purification steps or by expressing the recombinant protein in a deficient strain of cells.

This technique has been shown to be useful for following protein migration and trafficking and may be effective for measuring distance via Förster resonance energy transfer.

The fluorinated analog is incorporated into peptides via the relaxed substrate specificity of histidine-tRNA ligase and lowers the overall pKa of the tag.

[citation needed] The polyhistidine-tag can also be used for detecting a protein via anti-polyhistidine-tag antibodies, which can be useful for subcellular localization, ELISA, western blotting and other immuno-analytical methods.

Alternatively, in-gel staining of SDS-PAGE or native-PAGE gels with fluorescent probes bearing metal ions can be used for detection of a polyhistidine tagged protein.

A simple gravity flow column for Ni 2+ - affinity chromatography . The sample and subsequent buffers are manually poured into the column and collected at the bottom end after flowing through the resin bed (in light blue at the base of the column).
Three views of the X-ray structure of Ni(NTA)(H 2 O) 2 [ citation needed ]
Examples of methods for adding polyhistidine tags . ( A ) The polyhistidine tag is added by inserting the DNA encoding a protein of interest in a vector that has the tag ready to fuse at the C-terminus. ( B ) The polyhistidine tag is added using primers containing the tag coding sequence as an overhang on the forward primer. After PCR amplification, the tag is present on the N-terminus of the gene, which can then be sub-cloned into an expression vector.