Chinese Bulletin of Botany ›› 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

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

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

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