Abstract and subjects
Most cellular processes are driven by complex protein-protein interaction networks. Identifying key players and characterizing their interactions at the cellular and molecular level is of key importance to understand biochemical mechanisms that control cellular responses. Here, we detail a protocol for monitoring protein-protein interactions in E. coli cells or in cell extracts using a tripartite split-GFP system comprised of a protein interaction detector fragment (GFP beta-strands 1 through 9 or GFP1-9) and small tagging fragments of GFP beta-strands 10 (GFP10) and 11 (GFP11). Interaction of bait and prey proteins fused to GFP10 and GFP11 tether the small GFP fragments, allowing self-association with GFP1-9. In this scenario, fluorescence intensity of the reconstituted GFP is correlated with the strength of interaction.