Phospho-specific Immunoprecipitation and Detection

Total protein extract was processed for immunoprecipitation as described previously (Tomasi et al, 2012 (link)). Briefly, total cellular protein extract was pre-cleared with normal rabbit IgG (Santa Cruz) followed by 30 μl of protein A/G-agarose beads (SantaCruz). 200 Mg of pre-cleared protein was incubated with 2 μg of Pan-phospho antibody (Phos) overnight at 4°C under slow rotation. Immunoprecipitated protein was bound to 40 μl of protein A/G-agarose beads for 1 hour at 4°C under rotation and was washed five times with RIPA buffer containing protease inhibitors. After the final wash beads were heated at 95°C for 8 minutes in loading dye to elute the protein. Samples were processed for Western blotting as described above and developed with Clean-blot^™ IP detection reagent (HRP) (Thermo Scientific, Rockford, IL).