Protocol

Preparation of Clarified E. coli Extract Containing Histidine-Tagged Proteins

This protocol was adapted from "Immobilized Metal-Ion Affinity Chromatography," contributed by Lars Haneskog, Chapter 12, in Purifying Proteins for Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2004.

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INTRODUCTION

Histidine-tagged proteins can be isolated from cell extracts by passing them through an immobilized metal-ion affinity column (IMAC). The goals of extract preparation are to release the target protein from the cells and remove insoluble material that may foul the column filters and bed. This protocol describes a typical lysis method.

MATERIALS

Reagents

Escherichia coli cell culture harboring expressed recombinant histidine-tagged protein of interest

Imidazole-HCl (2 M, pH 7.4)

Imidazole purchased from Merck (Darmstadt, Germany) is essentially colorless. Other imidazole products on the market have been successfully used in IMAC, although many of them are discolored by impurities that affect the baseline . . . [Full Text of this Article]

METHOD