Protocol for determining protein cysteine thiol redox status using western blot analysis

• Published in: STAR protocols 2021-06, Vol.2 (2), p.100566-100566, Article 100566
• Main Authors: Pant, Bikram Datt, Oh, Sunhee, Mysore, Kirankumar S.
• Format: Article
• Language: English
• Subjects: Molecular Biology; Plant sciences; Protein Biochemistry; Protocol
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2021.100566

This protocol describes the analysis of protein cysteine redox status. Redox status is crucial in regulating protein activity, stability, and redox signaling cascades. It is determined by conjugation with 1.24 kDa MM(PEG)24 molecule to each reduced cysteine followed by western blot analysis. This protocol is easy to follow, and most of the reagents and instruments required are of common use in any lab. This protocol can be successfully applied to other biological sources. For complete details on the use and execution of this protocol, please refer to Pant et al. (2020). [Display omitted] •Protein redox status is crucial in regulating its activity, stability, and signaling•MM(PEG)24 reacts with reduced cysteine, increasing protein molecular weight by 1.24 kDa•Protein’s reduced status is determined by increased molecular weight in western blot This protocol describes the analysis of protein cysteine redox status. Redox status is crucial in regulating protein activity, stability, and redox signaling cascades. It is determined by conjugation with 1.24 kDa MM(PEG)24 molecule to each reduced cysteine followed by western blot analysis. This protocol is easy to follow, and most of the reagents and instruments required are of common use in any lab. This protocol can be successfully applied to other biological sources.