Cite as: Cold Spring Harb. Protoc.; 2010; doi:10.1101/pdb.prot5376

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Preparation of the Sample for Chromatofocusing: Buffer Exchange and Desalting

Jamil Mohammad

Adapted from Purifying Proteins for Proteomics (ed. Simpson). CSHL Press, Cold Spring Harbor, NY, USA, 2004.


INTRODUCTION

Chromatofocusing separates proteins on the basis of differences in their isoelectric point (pI). Successful chromatofocusing depends on careful preparation of the protein sample, as described in this protocol. The protein must be transferred to the same buffer that will be used to equilibrate the chromatofocusing column. The buffer pH should be higher than the pI of the protein to keep the proteins negatively charged, which facilitates their binding to the column. A protein sample containing particulate matter will decrease the resolution of the separation and shorten the column lifetime. Therefore, it is necessary to remove all insoluble and immiscible material from the sample prior to loading it on the column. The sample is first clarified by filtration or centrifugation and is then transferred into the start buffer using a high-capacity, fast-flowing, size-exclusion column packed with Sephadex G-25.


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