Summary
Immobilized metal affinity chromatography (IMAC) is a common place technique in modern protein purification. IMAC is distinct from most other affinity chromatography technologies in that it can operate on a native, unmodified protein without the need for a specialized affinity “tag” to facilitate binding. This can be particularly important where a protein of interest is to be separated from a complex mixture such as serum or an environmental isolate. Relying on the interaction of specific surface amino acids of the target protein and chelated metal ions, IMAC can provide powerful discrimination between small differences in protein sequence and structure. Additionally, IMAC supports have been demonstrated to function effectively as cation exchangers, allowing for two modes of purification with a single column. This chapter provides methodologies to perform IMAC in its most fundamental form, that of the interaction between histidine and immobilized metal ions, those that enable purification of proteins that lack surface histidines and the operation of IMAC supports in cation exchange mode.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Hemdan, E.S., Zhao, Y. J., Sulkowski, E. and Porath, J. (1989). Surface topography of histidine residues: A facile probe by immobilized metal ion affinity chromatography. Proc. Natl. Acad. Sci. U. S. A. 86, 1811–1815.
Wirth, H.-J., Unger, K.K. and Hearn, M.T.W. (1993). Influence of ligand density on the proteins of metal-chelate affinity supports. Anal. Biochem. 208, 16–25.
Porath, J., Carlsson, J., Olsson, I. and Belfrage, G. (1975). Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258, 598–599.
Everson, J.R., and Parker, H.E., (1974). Zinc binding and synthesis of 8-hydroxyquinoline-agarose. Bioinorg. Chem. 4, 15–20.
Ramadan, N., and Porath, J. (1985). Fe(III)hydroxamate as immobilized metal affinity-adsorbent for protein chromatography. J. Chromatogr. 321, 93–104.
Zachariou, M., and Hearn, M.T.W. (1996). Application of immobilized metal ion-chelate complexes as pseudocation exchange adsorbents for protein separation. Biochemistry 35, 202–211.
Zachariou, M., and Hearn, M.T.W. (1995). Protein selectivity in immobilized metal affinity chromatography based on the surface accessibility of aspartic and glutamic acid residues. J. Protein. Chem. 14, 419–430.
Beitle, R.R., and Ataali, M.M. (1992). Immobilized metal affinity chromatography and related techniques. AlChE Symposium Series 88, 34–44.
Wong, J.W., Albright, R.L. and Wang, N.-H. L. (1991). Immobilized metal ion affinity chromatography (IMAC) chemistry and bioseparation applications. Sep. Purif. Methods 20, 49–106.
Arnold, F.H. (1991). Metal-affinity separations: A new dimension in protein processing. BioTechnol. 9, 151–156.
Porath, J. (1992). Immobilized metal ion affinity chromatography. Protein Expr. Purif. 3, 263–281.
Sahni, S.K., and Reedijk, J. (1984). Coordination chemistry of chelating resins and ion-exchangers. Coord. Chem. Rev. 59, 1–139.
Pearson, R.G. (1990). Hard and soft acids and bases - The evolution of a chemical concept. Coordin. Chem. Rev. 100, 403–425.
Zachariou, M., and Hearn, M.T.W. (1992). High performance liquid chromatography of amino acids, peptides and proteins. CXXI. 8-hydroxyquinoline-metal chelate chromatographic support: an additional mode of selectivity in immobilized metal affinity chromatography. J. Chromatogr. 599, 171–177.
Zachariou, M., and Hearn, M.T.W. (1997). Characterization by potentiometric procedures of the acid-base and metal binding properties of two new classes of immobilized metal ion affinity adsorbents developed for protein purification. Anal. Chem. 69, 813–822.
Zachariou, M., and Hearn, M.T.W. (2000). Adsorption and selectivity characteristics of several human serum proteins with immobilised hard Lewis metal ion-chelate adsorbents. J. Chromatogr. 890, 95–116.
Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bateriophage T4. Nature 227, 680–685.
Amersham Biosciences (2003). Instructions 71–5001–87 AC: Chelating Sepharose Fast Flow.
Hansen, P., Lindeberg, G. and Andersson, L. (1992). Immobilized metal ion affinity chromatography of synthetic peptides. Binding via the alpha-amino group. J. Chromatogr. 215, 333–339.
Qiagen. (1998). The QIAexpressionist. A Handbook For high-Level Expression and Purification of 6xHis-Tagged Proteins, pp. 66.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Charlton, A., Zachariou, M. (2008). Immobilized Metal Ion Affinity Chromatography of Native Proteins. In: Zachariou, M. (eds) Affinity Chromatography. Methods in Molecular Biology™, vol 421. Humana Press. https://doi.org/10.1007/978-1-59745-582-4_2
Download citation
DOI: https://doi.org/10.1007/978-1-59745-582-4_2
Publisher Name: Humana Press
Print ISBN: 978-1-58829-659-7
Online ISBN: 978-1-59745-582-4
eBook Packages: Springer Protocols