The physiological concentrations of most proteins in the host are too low. To obtain large amounts of the proteins, their cDNAs are usually cloned in a suitable expression system. For purification using nickel affinity chromatography, a stretch of 6 histidine residues, known as 6×His tag, is incorporated at one of the protein termini. For nickel affinity chromatography, agarose or sepharose beads are functionalized with nitrilotriacetic acid (NTA). The NTA has four chelating sites for nickel ions thereby binding the ions very tightly. The Ni-NTA complex has very high affinity of the proteins having 6 consecutive histidine residues (Figure 25.1). The bound protein can be eluted using a high concentration of imidazole that competes with the histidine residues for the nickel ions. Ready to use Ni-NTA agarose and sapharose beads are commercially available.

Figure 25.1: Interaction of histidine residues in 6×His tag with Ni-NTA beads

Ni-NTA affinity chromatography can be performed as described in the protocol below wherein a Ni-NTA resin is packed into a column and cell lysate is loaded into the packed column. Alternatively, the Ni-NTA resin is mixed with the cell lysate and then column is packed with lysate-containing Ni-NTA resin (Batch purification, see Note 1).