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Immobilized Metal Ion Affinity Chromatography
of Native Proteins
Adam Charlton and Michael Zachariou
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.
Key Words: IMAC; protein purification; native protein; cation exchange.
1. Introduction
Immobilized metal affinity chromatography (IMAC) of proteins is a high
resolution liquid chromatography technique. It has the ability to differentiate a
single histidine residue on the surface of a protein (1), it can bind proteins with
dissociation constants of 10 −5 –10 −7 (2) and has had wide application in the
field of molecular biology for the rapid purification of recombinant proteins.
From: Methods in Molecular Biology, vol. 421: Affinity Chromatography: Methods and Protocols, Second Edition
Edited by: M. Zachariou © Humana Press, Totowa, NJ
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