The 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
The 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).