UNIT 9.4 Metal-Chelate Affinity Chromatography

  1. Kevin J. Petty

Published Online: 1 MAY 2001

DOI: 10.1002/0471140864.ps0904s04

Current Protocols in Protein Science

Current Protocols in Protein Science

How to Cite

Petty, K. J. 2001. Metal-Chelate Affinity Chromatography. Current Protocols in Protein Science. 4:9.4:9.4.1–9.4.16.

Author Information

  1. University of Texas Southwestern Medical Center, Dallas, Texas

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: JUN 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. This unit discusses techniques for creating a fusion protein consisting of the protein of interest with a histidine tail attached. A procedure for xpression of histidine-tail fusion proteins and their purification in native form by MCAC is described, and 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.