Cite as: Cold Spring Harb. Protoc.; 2010; doi:10.1101/pdb.prot5378

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Removal of Elution Buffer from Protein Sample Fractions after Chromatofocusing

Jamil Mohammad

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


INTRODUCTION

Chromatofocusing is a technique that separates proteins on the basis of differences in their isoelectric point (pI). Proteins are often eluted from a chromatofocusing column in Polybuffer or Pharmalyte. These buffers typically do not interfere with subsequent amino acid analysis or with the Coomassie blue protein assay. If buffer exchange and/or desalting is required, a number of methods are available to the researcher. Centrifugal filter units work well. As is described in this article, proteins can be concentrated and transferred to another buffer using ammonium sulfate precipitation which, in some cases, is performed in conjunction with hydrophobic interaction chromatography.


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