Immobilized Metal Ion Affinity Chromatography (IMAC)

Immobilized metal ion affinity chromatography (IMAC) is a rapid affinity purification chromatography. The proteins or peptides are separated based on their affinity for divalent metal ions chelated to a solid matrix such as beaded agarose or magnetic beads (Fig1.). At pH 7-8, protein or peptide containing consecutive amino acids such as histidine, tryptophan, and cysteine will bind to the chelated metal ions (e.g., Zn2+, Cu2+, Cd2+, Hg2+, Co2+, Ni2+, and Fe2+). Iminodiacetate (IDA) and nitrilotriacetic acid (NTA) are commonly chelating groups, and Ni2+ and Co2+ are the mainly used ions. The structure of the ligand IDA is shown in Figure 1. The binding reaction with the target protein is affected by various independent variables such as pH, temperature, salt type, salt concentration, immobilized metal and chelate ligand density, and protein size. The bound protein is eluted by a decreasing pH gradient, by an increasing imidazole concentration, or by adding an EDTA chelator in a buffer. This technique is an ideal tool for capturing and purification of his-tagged recombinant protein in a quick, inexpensive, and straightforward manner.