GFP-trap pulldowns

All steps were performed at 4°C. HEK 293FT transfected cells were collected after 48 hr, washed 3 times with 1x PBS and lysed in 1 ml of lysis buffer [25 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% Triton X-100, 5% glycerol, protease inhibitors (Roche)]. Lysates were kept on ice for 30 min vortexing every 5 min and spun down at 25,000 x g for 20 min. After saving 40 μl of supernatant (input), the rest was incubated overnight with 30 μl of pre-washed GFP-Trap resin (Chromotek) in a rotating wheel. Beads were washed 5 times for 5 min each with WB (25 mM Tris-HCl pH 7.5, 300 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% TX-100, 5% glycerol). Beads were centrifuged at 2000 x g for 2 min after each wash. For elution, samples were boiled for 5 min at 95°C in 2x Laemmli buffer.