Chin Bull Bot ›› 2019, Vol. 54 ›› Issue (4): 497-502.doi: 10.11983/CBB19108

• TECHNIQUES AND METHODS • Previous Articles     Next Articles

Detection and Analysis of Protein S-nitrosylation in Plants

Chen Lichao,Zhan Ni,Li Yansha,Feng Jian,Zuo Jianru*()   

  1. Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
  • Received:2019-06-13 Accepted:2019-06-20 Online:2020-01-08 Published:2019-07-01
  • Contact: Zuo Jianru E-mail:jrzuo@genetics.ac.cn

Abstract:

S-nitrosylation is an important protein posttranslational modification, involved in covalently linking a nitric oxide (NO) molecule to the thiol group of a cysteine residue to generate S-nitrosothiols. S-nitrosylation regulates multiple biological processes by modulating protein activity, stability, subcellular localization and protein-protein interactions. The biotin-switch assay is one of the most-often used methods to detect and analyze protein S-nitrosylation. In principle, the free thiols in a target protein are first blocked, followed by reducing the S-nitrosothiols of the target protein to free thiols by ascorbate, which are subsequently labelled by biotin to form biotinylated proteins. The biotin-labelled sample was assayed by immunoblotting and mass spectrometry. Here, we present detailed experimental procedures for the in vitro and in vivo biotin-switch methods and give advice on key troubleshooting solutions.

Key words: nitric oxide, S-nitrosylation, biotin-switch assay, plants

Figure 1

In vitro analysis of S-nitrosylated bovine serum albumin (BSA) BSA is labelled with biotin-maleimide, subjected to SDS- PAGE and western blotting. Anti-biotin and anti-BSA antibodies are used as primary antibodies, respectively (dilution in 1:20 000) and anti-mouse IgG as a secondary antibody. Signals are detected by using the SuperSignal Western Femto Maximun Sensitivity Substrate Kit. The blots are exposed for 20 sec (top) and 30 sec (bottom), respectively. GSH: Glutathione; GSNO: S-nitrosoglutathione; Asc: Sodium ascorbate"

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