The oxidation and reduction of cysteine residues is emerging as an important post-translational control of protein function. We describe a method for fluorescent labelling of either reduced or oxidized thiols in combination with two-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (2DE) to detect changes in the redox proteome of cultured cells. Reduced thiols are labelled with the fluorescent compound 5-iodoacetamidofluorescein. To monitor oxidized thiols, the reduced thiols are first blocked with N-ethyl-maleimide, then the oxidized thiols reduced with dithiothreitol and labelled with 5-iodoacetamidofluorescein. The method is illustrated by treating Jurkat T-lymphoma cells with hydrogen peroxide and monitoring increased labelling of oxidized thiol proteins. A decrease in labelling can also be detected, and this is attributed to the formation of higher oxidation states of cysteine that are not reduced by dithiothreitol.
CITATION STYLE
Cuddihy, S. L., Baty, J. W., Brown, K. K., Winterbourn, C. C., & Hampton, M. B. (2009). Proteomic detection of oxidized and reduced thiol proteins in cultured cells. Methods in Molecular Biology (Clifton, N.J.), 519, 363–375. https://doi.org/10.1007/978-1-59745-281-6_23
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