Anti flag m2 affinity gel 50 slurry

Cells transfected with plasmid DNA were lysed in RIPA buffer with the addition of protease inhibitor and phosphatase inhibitor (Roche). The cell extracts were harvested by centrifugation at 4°C for 10 min and the supernatant was used for IP assay. Cell extracts (500 μg) were incubated with 20 μl of anti-Flag M2 affinity gel (50% slurry) (Sigma-Aldrich, St. Louis, MO) at 4°C for 2 h. Immunoprecipitates were collected by centrifugation at 1000 × g, washed with RIPA buffer for three times followed by SDS-PAGE electrophoresis and western blot. For co-IP assay, cells were co-transfected with equal amount of Tag-fusioned constructs and lysed 48 h later. anti-Flag M2 affinity gel (50% slurry) (Sigma-Aldrich) and EGFP antibody (Roche) were used for IP of protein complex from cell extract at 4°C for 2 h. After washing with PBS for three times, the immunoprecipitates were eluted by sampling buffer and subject to SDS-PAGE and western blot.