Glutathione sepharose 4 fast flow beads

To express recombinant His-GI, GST-SOS1^453–1146, and GST proteins, Escherichia coli BL21 (DE3) star cells were transformed with pHis-SUMO-GI, pGEX4T-SOS1^453–1146, and pGEX-2T, respectively. The transformed cells were grown at 37 °C (optical density₆₀₀ = 0.8). Protein expression was induced by treating the cells with 0.5 mM isopropyl-1-thio-β-_D-galactopyranoside (IPTG) for 4 h at 30 °C for GST-SOS1^453–1146 and GST or 0.1 mM IPTG for 24 h at 15 °C for His-GI. Recombinant proteins were extracted as described previously (5 (link)). Supernatants containing GST-SOS1^453–1146 or GST were immobilized onto Glutathione Sepharose 4 Fast Flow beads (Cytiva) for 2 h at 4 °C, and the beads were washed with cold 1x phosphate-buffered saline (PBS). His-GI protein was purified in Ni-NTA Agarose (Qiagen) using 250 mM imidazole in 1x PBS. GST-SOS1^453–1146– or GST-bound beads were further incubated with purified His-GI in cold GST lysis buffer (55 ) for 2 h at 4 °C with rotation. The beads were washed five times with cold GST lysis buffer, resuspended in SDS-PAGE sample buffer, briefly heated at 95 °C for 3 min, and subjected to SDS-PAGE. His-GI bound to GST-SOS1^{453 − 1146} or GST was validated by immunoblot analysis using anti-His antibody (1:500, Thermo Fisher Scientific).

GST and GST-LC3 fusion proteins were incubated with glutathione-Sepharose 4 Fast Flow beads (Amersham Biosciences, 20182003–2) at 4 °C for 2 h and then washed 3 times with 1 ml ice-cold PBS. After the indicated treatments or transfections, cell lysates were prepared and subsequently added to the conjugated beads, followed by incubation at 4 °C overnight. The precipitated complexes were washed five times with 1 ml ice-cold PBS and boiled with loading buffer at 100 °C for 3 min and analyzed via SDS-PAGE and western blotting using specific antibodies. The GST and GST-LC3 bands on the membrane were visualized through Ponceau S staining.