Cite as: Cold Spring Harb. Protoc.; 2006; doi:10.1101/pdb.prot4264

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Purification of Histidine-Tagged Proteins Using Stepwise Elution with Imidazole

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 15% of the full text of this article appears below.


INTRODUCTION

Purification of histidine-tagged protein using gradient elution from an immobilized metal-ion affinity chromatography (IMAC) column can be performed without prior optimization. However, determining an optimized gradient elution (as per Optimization of an Imidazole Gradient for Purification of Histidine-Tagged Proteins) enables a histidine-tagged protein to be purified using stepwise elution. This protocol provides a small-scale procedure for ensuring that purification of the desired protein is satisfactory.


MATERIALS

Reagents

recipe IMAC binding buffer

recipe IMAC high imidazole buffer

Protein . . . [Full Text of this Article]

Equipment


METHOD


TROUBLESHOOTING


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