Anti gfp antibody conjugated agarose beads

Exogenous RhoA-GFP was overexpressed in cells treated with siVCP, siLuc (negative control) and siLuc with MG132 treatment (positive control). Cells were harvested with Pierce IP-lysis buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 5% glycerol). RhoA-GFP from different samples was then immunoprecipitated by anti-GFP antibody conjugated agarose beads (ChromoTek) followed by western blot analysis using anti-ubiquitin antibody (Santa Cruz).

GFP-tagged p97/VCP expressing cells were harvested with Pierce IP-lysis buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 5% glycerol) supplemented with protease and phosphatase inhibitors. Lysates were incubated with anti-GFP antibody conjugated agarose beads or unconjugated agarose beads (Chromotek) for four hours at 4°C. The immunoprecipitants were then washed three times and heated in sample buffer for downstream analyses. In western blot analysis, the resultant SDS-PAGE gel was transferred onto a nitrocellulose membrane and probed with the relevant antibodies. Each immunoblot experiment was repeated at least three times for consistency. Densitometric analyses were performed using ImageJ.