Immuno-Spin Trapping Method for the Analysis of S-Nitrosylated Proteins.
DMPO
S-nitrosylation
immune-spin trapping
thiols
thiyl radical
Journal
Current protocols
ISSN: 2691-1299
Titre abrégé: Curr Protoc
Pays: United States
ID NLM: 101773894
Informations de publication
Date de publication:
Sep 2021
Sep 2021
Historique:
entrez:
27
9
2021
pubmed:
28
9
2021
medline:
30
9
2021
Statut:
ppublish
Résumé
The dynamic and unstable nature of protein nitrosothiols (PSNOs) derived from complex biological matrices (like cell lysates) make them unsuitable for proteomic/biochemical analysis in vitro. In an attempt to increase the stability of cell-derived PSNOs, scientists have devised methods to derivatize thiols undergoing nitrosylation, with a suitable molecule, to yield a stable adduct that could easily be detected using appropriate antibodies. The Biotin Switch Assay (BTSA) is currently the most widely used method for tagging PSNOs; however, the error-prone and cumbersome nature of the BTSA protocol prompted the development of alternative mechanisms of tagging cell-derived PSNOs. One such method is the immuno-spin trapping method using 5,5-dimethyl-1-pyrroline N-oxide (DMPO), which effectively overcomes the shortcomings of the BTSA and proves to be a promising alternative. Here we describe the protocol for DMPO-based PSNO labeling and subsequent proteomic analysis by western blotting with an anti-DMPO antibody. © 2021 Wiley Periodicals LLC. Basic Protocol: Labeling of cell-derived PSNOs by DMPO-based immuno-spin trapping and their subsequent analysis by immunostaining.
Substances chimiques
Free Radicals
0
Proteins
0
Sulfhydryl Compounds
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
e262Informations de copyright
© 2021 Wiley Periodicals LLC.
Références
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