Proteomics/Protein Separations - Chromatography/Immobilized Metal Ion Affinity Chromatography (IMAC)

Chapter written by: Laura Grell and Alexander Butarbutar
Contact llg3875@rit.edu or nbb3924@rit.edu for contributions
Chapter modified by Kai Burnett and Dalia Ghoneim
Contact kab9783@rit.edu or dxg6098@rit.edu

Immobilized Metal Ion Affinity Chromatography (IMAC)

   Immobilized metal ion/Metal Chelate affinity chromatography is a separation technique that is based on coordinate covalent binding between proteins and metal ions. Proteins have a wide variety of amino acids composition which, in effect, generates a range of different affinities towards metal ions. However, not many naturally occurring proteins have affinity for metal ions, so the technique is mainly used to purify recombinant proteins. For example, proteins can be engineered to contain a poly-histidine tail (histidine can generally act as a ligand towards divalent metal cations). If the stationary phase is immobilized with divalent metal cations, a mixture of proteins can be separated based on their ability to interact with the metal ions. Those proteins containing a higher number of histidine residues would be able to bind to the column more tightly than those with fewer histidine residues.

   Several different types of immobilized metal ion column have been developed to separate various proteins (e.g. Fe, Co, Cd, Ni, or Zn). Protein separation in IMAC generally depends on the strength of the metal ion-protein bond. Thus, choosing the type of immobilized ion is crucial to successful protein separation. By far the most widely-used technique is to use an immobilized nickel column, and to engineer poly-histidine tags of six or more residues onto the recombinant proteins of interest. One thing to keep in mind is that the binding between metal ion and protein must be reversible, allowing elution of bounded protein at later steps. Three different elution strategies can be applied to IMAC competitive elution, stripping elution and pH Adjustment (Ref1).

< Previous Page | Next Page >

Resources

1. Affiland Immobilized metal ion affinity chromatography (IMAC)
2. GE Healthcare Immobilized metal ion affinity chromatography products
3. Merck Metal Chelate Affinity Chromatography Using Tentacle Gels
4. Bio-Rad Profinity IMAC Resins

References

1. Paunovic. I, Schulin. R, Nowack, B. Evaluation of immobilized metal-ion affinity chromatography for the fractionation of natural Cu complexing ligands Journal of Chromatography A, 1100 (2005) 176–184. *
2. Yip TT, Hutchens TW. Immobilized metal-ion affinity chromatography. Methods Mol Biol. 2004;244:179-90.
3. Sun X, Chiu JF, He QY.Application of immobilized metal affinity chromatography in proteomics. Expert Rev Proteomics. 2005 Oct;2(5):649-57. *
4. Zachariou M. Immobilized metal ion affinity chromatography of proteins. Methods Mol Biol. 2004;251:89-102.
5. Belew M, Porath J. Immobilized metal ion affinity chromatography. Effect of solute structure, ligand density and salt concentration on the retention of peptides. J Chromatogr. 1990 Sep 21;516(2):333-54.

.* Denotes Free Article