Abstract
Nitric oxide (NO) plays a pivotal role in cellular signaling in many different organisms as the result of the modification of protein activities/functions by protein S-nitrosylation. This NO-dependent posttranslational modification is based on the attachment of NO to the sulfur moiety of cysteine residues. However, the instability of S-nitrosothiols makes it difficult to analyze this type of protein modification in vitro as well as in vivo. Jeffrey and colleagues developed a method—named the biotin switch method—that allows the detection and purification of S-nitrosylated proteins. The principle behind this technology is the substitution of the NO group by a biotin linker in a three-step procedure. First, the all free thiol groups are blocked with a thiol-reactive agent, followed by selective reduction of the S-nitrosylated cysteine residues using ascorbate. In the final step, the reduced thiol groups are labeled with a biotin linker, so that the previously S-nitrosylated cysteine residues are finally biotinylated. Afterwards, the biotinylated proteins can be detected with anti-biotin antibodies or can be purified by affinity chromatography on neutravidin agarose. In this chapter, we give a detailed description of the biotin switch method, which can be used for proteomics approach to identify candidates for protein S-nitrosylation as well as to analyse S-nitrosylation of selected proteins.
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Lindermayr, C., Sell, S., Durner, J. (2008). Generation and Detection of S-Nitrosothiols. In: Hancock, J.T. (eds) Redox-Mediated Signal Transduction. Methods in Molecular Biology™, vol 476. Humana Press. https://doi.org/10.1007/978-1-59745-129-1_15
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DOI: https://doi.org/10.1007/978-1-59745-129-1_15
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