Immobilized metal ion affinity chromatography of native proteins.
Immobilized metal affinity chromatography (IMAC) is a common place technique in modem 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.
Charlton
Adam
A
Industrial Biotechnology, CSIRO Molecular and Health Technology, Australia.
Zachariou
Michael
M
D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D008670:Metals / Q000737:chemistry*; D009994:Osmolar Concentration; D011506:Proteins / Q000302:isolation & purification*
D008670:Metals; D011506:Proteins