Protein-Protein Interaction Assays: Co-IP and GST-Pulldown

Co-immunoprecipitation assays and GST-pulldown assay were applied for detection of protein–protein interaction. Cells were lysed with 1 ml lysis buffer (containing 20 mM HEPES (pH 7.4), 1% Triton-100, 100 mM NaCl, 5 mM MgCl2, and EDTA-free proteinase inhibitor cocktail from Roche) for 1 h on ice. The cell lysates were clarified by spinning at 14,000 rpm for 30 min.
For co-immunoprecipitation assay, INS-1 cells lysates were immunoprecipitated with anti-Syt1 antibody. For GST-pulldown assay, 293T cells transfected with the indicated plasmids. The supernatants were incubated with GST-fusion protein coupled to GST-Sepharose 4B resin (GE Healthcare, cat. No. 45-000-139) at 4°C for 4 h. The proteins bound to GST sepharose beads were detected through western blot assay.
Western blot was performed as described [58 (link)]. Briefly, cells were lysed in RIPA buffer containing proteinase inhibitor cocktail (Roche, cat. no. 04693132001). The resulted cell lysates were separated by SDS-PAGE, the resolved proteins were transferred to PVDF membrane, and then the membrane was blocked with 5% milk in TBST, followed by incubation with primary antibodies and HRP-conjugated secondary antibody. The blots were detected using ECL system (Pierce, Rockford, IL, USA).