Knowledge of the interaction of metal ions with proteins and the potential utility of immobilized metal chelators began during 1940-50, although it was not until 1974 that the method was first used to isolate a metalloprotein. The general use of IMAC was initiated in 1975 with a Nature publication from Porath. A summary of key milestones in the history of IMAC is presented in Table 1.

In the late 1970s and 1980s there were numerous publications on the choice of metals and investigations on the precise nature of the interactions that take place with proteins. It was assumed that surface-exposed residues were coordinating with the immobilized metal ions. Studies using free amino acids, peptides, and eventually engineered recombinant proteins, revealed the importance of certain amino acids

Liu X-C and Scouten WH (1996) Studies on oriented and reversible immobilization of glycoprotein using novel boronate affinity gel. Journal of Molecular Recognition, 9: 462-467.

West I and Goldring O (1996) Lectin affinity chromatography. In: Doonan S (ed.) Methods in Molecular Biology, Protein Purification Protocols, vol. 59, pp. 177-185. New Jersey: Humana Press.

- in particular histidine. Additionally, depending on the metal and chelating ligand employed, the spatial arrangement of the amino acids within the peptide or protein was also found to influence binding. This led to studies using model peptides with a wide range of histidine-containing sequences and in 1988 the first use of six consecutive histidine-residues as a purification tag (6His tag). In parallel with this 1987 saw the introduction of a metal-chelate complex with a high degree of selectivity for adjacent histidine residues (Ni2+-NTA). Proteins purified using the 6His tag have been found to retain biological activity and their structures have also been solved by both X-ray and NMR - illustrating that, in the absence of metal ions, the tag has no defined secondary structure. Despite the enormous utility of the 6His tag the use of metal chelating ligand/metal combinations still has a role to play in the isolation of nontagged proteins from a wide variety of sources. The literature contains many examples of using IMAC as a one-step process to isolate native proteins, e.g. a-lactalbumin from milk and factor IX from blood. In addition, immobilized Fe3+ has been successfully used to separate phosphoproteins and immobilized Ca2+ to purify calcium-binding proteins.

The potential exists for even wider application to the separation of protein mixtures, with new chelators being introduced (e.g. TACN, see below).

Table 1 Key dates in the history of immobilized metal ion affinity chromatography


First use of immobilized chelators to isolate



First description of general technique (IMAC)

using IDA


Introduction of high performance on silica based



Use of Fe3+ chelates to purify phosphoproteins


Introduction of NTA


Introduction of genetically engineered His tags


Introduction of TREN


Introduction of TACN

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