Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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Purification of Polyhistidine-Tagged Proteins by Immobilized Metal Affinity Chromatography

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Immobilized metal affinity chromatography (IMAC) is based on the affinity of polyhistidine tracts for divalent metal cations (usually Ni2) immobilized as transition metal chelate complexes on a chromatography resin (for review, see Bornhorst and Falke 2000; Block et al. 2009). Because few natural proteins bind to immobilized divalent metal ions with significant affinities, recombinant polyhistidine-labeled proteins can be purified to near homogeneity, often in a single step, using metal chelate affinity chromatography. Wash steps remove the majority of contaminating proteins that bind the resin, and the protein of interest is eluted with a soluble competing chelator. General considerations during IMAC include accessibility of the polyhistidine tag, type of metal ion and chelating matrix, and the pH and buffer components for binding, wash, and elution steps. Metal affinity chromatography is popular because it is both highly effective and relatively insensitive to protein folding, ionic strength, chaotropes, and detergents. Because of its high binding capacity, up to 50 mg of recombinant protein can be purified using only 1 mL of IMAC resin (2.5 mol of a 20-kDa protein using Ni2NTA agarose; QIAGEN). Considering the high efficiency, capacity, concentrating power, and speed of this technique, it is often the first (and sometimes only) strategy used when developing a purification protocol. Details of IMAC mechanics are presented in the Discussion section.


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(Limited time special offer.) Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook
Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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