Please cite as: CSH Protocols; 2006; doi:10.1101/pdb.prot4263

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Optimization of an Imidazole Gradient for Purification of Histidine-Tagged Proteins

Lars Haneskog

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.

The first 100 words of the full text of this article appear below.


INTRODUCTION

Purification using gradient elution can be performed without prior optimization, because most contaminants bound to the immobilized metal-ion affinity chromatography (IMAC) column will elute at imidazole concentrations different from those of the histidine-tagged protein. However, a gradient elution test run can be performed to determine suitable imidazole concentrations for the wash and elution steps, so that an optimized purification protocol with stepwise elution can be established (see Purification of Histidine-Tagged Proteins Using Stepwise Elution with Imidazole).


MATERIALS

Reagents

recipe IMAC binding buffer

recipe IMAC high imidazole buffer

Protein extract prepared as in Preparation of Clarified E. coli Extract Containing Histidine-Tagged Proteins

Equipment

Chromatography system . . . [Full Text of this Article]


METHOD


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