Detection of protein-protein interactions using the GST fusion protein pull-down technique

Glutathione-S-transferase (GST) fusion proteins have had a range of applications since their introduction as tools for synthesis of recombinant proteins in bacteria¹. Typically, GST pull-down experiments are used to identify interactions between a probe protein and unknown targets and to confirm suspected interactions between a probe protein and a known protein^{2, 3}. The probe protein is a GST fusion, whose coding sequence is cloned into an isopropyl-â-D-thiogalactoside (IPTG)-inducible expression vector. This fusion protein is expressed in bacteria and purified by affinity chromatography on glutathione-agarose beads. Target proteins are usually lysates of cells, either labeled with [³⁵S]methionine or unlabeled, depending on the method used to assay the interaction between the target and the probe. The cell lysate and the GST fusion protein are incubated together with glutathione-agarose beads. Complexes recovered from the beads are resolved by SDS-PAGE and analyzed by western blotting, autoradiography or staining.