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About thiol derivatization and resolution of basic proteins in two‐dimensional electrophoresis
Author(s) -
Luche Sylvie,
Diemer Hélène,
Tastet Chistophe,
Chevallet Mireille,
Van Dorsselaer Alain,
LeizeWagner Emmanuelle,
Rabilloud Thierry
Publication year - 2004
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200300589
Subject(s) - thiol , derivatization , resolution (logic) , chemistry , maleimide , cysteine , streaking , disulfide bond , electrophoresis , gel electrophoresis , chromatography , combinatorial chemistry , biochemistry , organic chemistry , high performance liquid chromatography , biology , enzyme , computer science , artificial intelligence , microbiology and biotechnology
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.