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Generation and detection of S-nitrosothiols.
In: Redox-Mediated Signal Transduction. Berlin [u.a.]: Springer, 2008. 210-222 (Methods Mol. Biol. ; 476)
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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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Publikationstyp Artikel: Sammelbandbeitrag/Buchkapitel
Herausgeber Hanock, J.T.*
Schlagwörter Biotin switch; S-nitrosylation; nitric oxide
ISSN (print) / ISBN 1064-3745
Bandtitel Redox-Mediated Signal Transduction
Zeitschrift Methods in Molecular Biology
Quellenangaben Band: 476, Seiten: 210-222
Verlagsort Berlin [u.a.]
Begutachtungsstatus Peer reviewed
Institut(e) Institute of Biochemical Plant Pathology (BIOP)