Protein Purification Protocols

Volume 244 of the series Methods in Molecular Biology pp 179-190

Immobilized Metal-Ion Affinity Chromatography

  • Tai-Tung YipAffiliated withCiphergen Biosystems Inc.
  • , T. William HutchensAffiliated withLumiCyte Inc.

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Immobilized metal-ion affinity chromatography (IMAC) (14) is also referred to as metal chelate chromatography, metal-ion interaction chromatography, and ligand-exchange chromatography. We view this affinity-separation technique as an intermediate between highly specific, high-affinity bioaffinity separation methods, and wider-spectrum, low-specificity adsorption methods, such as ion exchange. The IMAC stationary phases are designed to chelate certain metal ions that have selectivity for specific groups (e.g., His residues) in peptides (e.g., refs. 59) and on protein surfaces (1015). The number of stationary phases that can be synthesized for efficient chelation of metal ions is unlimited, but the critical consideration is that there must be enough exposure of the metal ion to interact with the proteins, preferably in a biospecific manner. Several examples are presented in Fig. 1. The challenge to produce new immobilized chelating groups, including protein surface metal-binding domains (17,18) is being explored continuously 19). A common fusion protein is the hexahistidine tag for purification (20). Table 1 presents a list of published procedures for the synthesis and use of stationary phases with immobilized chelating groups. This is by no means exhaustive and is intended only to give an idea of the scope and versatility of IMAC.
Fig. 1.

Schematic illustration of several types of immobilized metal-chelating group, including iminodiacetate (IDA), tris(carboxymethyl) ethylenediamine (TED), and the metal-binding peptides (GHHPH) n G (where n=1,2, 3, and 5. (From refs. 17 and 18.)

Table 1

Immobilized Chelating Groups and Metal Ions Used for Immobilized Metal-Ion Affinity Chromatography

Chelating group

Suitable metal ions


Commercial source a



(1, 2)

Pharmacia LKB Pierce Sigma Boehringer-Mannheim TosoHaas




Not available

α-Alkyl nitrilotriacetic acid



Not available

Carboxymethylated aspartic acid



Not available




Not available

(GHHPH) n G b


17, 18

Not available

a Locations: Pharmacia LKB, Uppsala, Sweden; Pierce, Rockford, IL; Sigma, St. Louis, MO; Boeh-ringer-Mannheim, Mannheim, Germany; TosoHaas, Philadelphia, PA.

b Letters represent standard one-letter amino acid codes (G = glycine; H = histidine; P = proline). The number of internal repeat units is given by n (n = 1, 2, 3, and 5).