Glutathione S-transferase pull-down assay.

Abstract

AbstractThe glutathione S-transferase (GST) pull-down assay is a relatively easy, straightforward method to identify potential protein kinase C (PKC)-binding partners. The method is also extensively used to confirm known interactions and to map interaction sites. The pull-down method relies on the immobilization of a GST fusion protein on glutathione sepharose beads that serve as a solid phase. The first step requires the expression of the PKC domain of interest as a fusion protein with the GST moiety. After binding of the GST fusion protein to the glutathione sepharose matrix, the mixture is incubated with whole-cell homogenate or a purified protein. Nonbound material is washed off the column, and subsequently the binding complex is eluted. Upon elution, the mixture is resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Coomassie staining, silver staining, or Western blot. This chapter focuses on the GST tag; however, other tags, such as hexa-Histidine (6xHis) and maltose-binding protein (MBP), are also commonly used and will be mentioned throughout the chapter (see Note 1).KeywordsGlutathione Sepharose BeadWash BeadPotential Protein KinaseDiagnostic CorporationStore PowderThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.