UNIT 10.11B Metal-Chelate Affinity Chromatography
Published Online: 1 MAY 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Molecular Biology
How to Cite
Petty, K. J. 2001. Metal-Chelate Affinity Chromatography. Current Protocols in Molecular Biology. 36:IV:10.11B:10.11.10–10.11.24.
- Published Online: 1 MAY 2001
- Published Print: OCT 1996
Recombinant proteins engineered to have six consecutive histidine residues on either the amino or carboxyl terminus can be purified using a resin containing nickel ions (Ni2+) that have been immobilized by covalently attached nitrilotriacetic acid (NTA). This technique, known as metal-chelate affinity chromatography (MCAC), can readily be performed with either native or denatured protein. Techniques are discussed for creating a fusion protein consisting of the protein of interest with a histidine tail attached (for purification by MCAC). The basic protocol describes expression of histidine-tail fusion proteins and their purification in native form by MCAC. Two alternate protocols describe purification of histidine-tail fusion proteins by MCAC under denaturing conditions and their renaturation by either dialysis or solid-phase renaturation. Support protocols are provided for analysis of the purified product and regeneration of the NTA resin. All of these protocols are easily adaptable to any protein expression system.