Summary
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.
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Acknowledgments
The authors would like to thank Juliet Pullar, Andrew Cox, and Rachel Wilkie for valuable discussions during method development. Financial support has come from the Royal Society Marsden Fund and the Health Research Council of New Zealand. Sarah Cuddihy is supported by an FRST Post-Doctoral Fellowship and Kristin Brown has a TEC Top Achievers Doctoral Scholarship.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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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. In: Tyther, R., Sheehan, D. (eds) Two-Dimensional Electrophoresis Protocols. Methods in Molecular Biology, vol 519. Humana Press. https://doi.org/10.1007/978-1-59745-281-6_23
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DOI: https://doi.org/10.1007/978-1-59745-281-6_23
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