Your browser doesn't support javascript.
loading
Immunoprecipitation methods to identify S-glutathionylation in target proteins.
Butturini, Elena; Boriero, Diana; Carcereri de Prati, Alessandra; Mariotto, Sofia.
Afiliação
  • Butturini E; Department of Neuroscience, Biomedicine and Movement Sciences, Section of Biological Chemistry, University of Verona, Verona, Italy.
  • Boriero D; Department of Neuroscience, Biomedicine and Movement Sciences, Section of Biological Chemistry, University of Verona, Verona, Italy.
  • Carcereri de Prati A; Department of Neuroscience, Biomedicine and Movement Sciences, Section of Biological Chemistry, University of Verona, Verona, Italy.
  • Mariotto S; Department of Neuroscience, Biomedicine and Movement Sciences, Section of Biological Chemistry, University of Verona, Verona, Italy.
MethodsX ; 6: 1992-1998, 2019.
Article em En | MEDLINE | ID: mdl-31667096
S-glutathionylation is a reversible post-translational modification of proteins that generate a mixed disulfide between glutathione to thiolate anion of cysteine residues in target proteins. In the last ten years, S-glutathionylation has been extensively studied since it represents the cellular response to oxidative stress, in physiological as well as pathological conditions. This modification may be a protective mechanism from irreversible oxidative damage and, on the other hand, may modulate protein folding and function. Due to the importance of S-glutathionylation in cellular redox signaling, various methods have been developed to identify S-gluthationylated proteins. Herein, we describe two easy methods to recognized S-glutathionylation of a target protein after oxidative stress in cellular extracts based on different immunoprecipitation procedures. The immunoprecipitation assay allows the capture of one glutathionylated protein using a specific antibody that binds to the target protein. The presence of S-glutathionylation in the immunoprecipitated protein is identified using anti-glutathione antibody. The second type of approach is based on the detection of the glutathionylated protein with biotin/streptavidin technique. After different steps of protection of non-oxidized thiolic groups and reduction of S-glutathionylated groups, the newly-formed protein free-thiols are labeled with biotin-GSH. The modified protein can be isolate with streptavidin-beads and recognized using an antibody against target protein. •S-glutathionylation is a reversible post-translational modification of proteins that recently has been emerged as important signaling in the redox regulation of protein function.•Both methods to identify glutathionylated proteins are economic, easy and do not require particular equipment.•The setups of both methods guarantee high reproducibility.
Palavras-chave

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Idioma: En Ano de publicação: 2019 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Idioma: En Ano de publicação: 2019 Tipo de documento: Article