GFP-TRAP Immunoprecipitation

16HBE cells expressing GFP-tagged proteins were cultured on 10 cm dishes until 100% confluent. Cells were then washed three times with ice-cold PBS before 500 µl of cold GFP-trap buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP40, 15 mM MgCl₂, 10% glycerol, 1% Triton X-100, 5 mM EDTA containing protease and phosphatase inhibitor cocktails (1:100) was added. Cells were subsequently scraped and centrifuged at 5000 × g for 10 min at 4 °C. 1:1 of GFP-trap beads and control agarose resin was washed three times with IP lysis buffer. Supernatants obtained from cell lysates were added to the beads and incubated on a rotator at 4 °C for 2 h. 50 µl of supernatants were also set aside as input. Afterwards, the beads were washed three times with lysis buffer. 2× sample buffer containing β-mercaptoethanol (1:100) was added to the beads, boiled for 5 min at 95 °C and centrifuged to clear cell debris. 40 μl of samples were loaded and then subjected to Western blot analysis.