Cite as: Cold Spring Harb. Protoc.; 2010; doi:10.1101/pdb.prot5375

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Preparation of the Gel and Column for Chromatofocusing

Jamil Mohammad

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


INTRODUCTION

The appropriate stationary phase for chromatofocusing depends very much on the intended application. In general, in order to form a linear pH gradient (which is critical for good separation), the stationary phase must provide uniform buffering capacity over the pH separation range of interest. Thus, the functional groups on the matrix must be able to provide high buffering capacity over a broad pH range. Examples of such functional groups are polyethyleneimine (PEI) and a mixture of tertiary and quaternary amines. The most commonly used stationary phases for chromatofocusing are polymer-based stationary phases such as Mono P or polybuffer exchanger (PBE). In this protocol, a column is readied and packed with PBE matrix. The matrix is equilibrated with the buffer that will be used at the start of the separation, and the quality of the packing is assessed using cytochrome c.


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