About thiol derivatization and resolution of basic proteins in two-dimensional electrophoresis

Abstract

The influence of thiol blocking on the resolution of basic proteins by two-dimensional electrophoresis was investigated. Cysteine blocking greatly increased resolution and decreased streaking, especially in the basic region of the gels. Two strategies for cysteine blocking were found to be efficient: classical alkylation with maleimide derivatives and mixed disulfide exchange with an excess of a low molecular weight disulfide. The effect on resolution was significant enough to allow correct resolution of basic proteins with in-gel rehydration on wide gradients (e.g. 3-10 and 4-12), but anodic cup-loading was still required for basic gradients (e.g. 6-12 or 8-12). These results demonstrate that thiol-related problems are not solely responsible for streaking of basic proteins on two-dimensional gels.

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References in corpus (2)

• Improvement of the solubilization of proteins in two-dimensional electrophoresis with immobilized pH gradients
• Progress in the definition of a reference human mitochondrial proteome

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