Mouse monoclonal anti rnf126

Total cell extracts were prepared in ice-cold modified immunoprecipitate (IP) buffer (25 mM Tris-HCl [pH 7.5], 125 mM NaCl, 1% glycerol, 1 mM MgCl₂, 0.5% Triton, 0.5% Igepal CA-630, 0.5% sodium deoxycholate) supplemented with complete protease inhibitor cocktail (Roche), 5 mM NEM, 1 mM sodium orthovanadate (NaV), and 5 mM sodium fluoride (NaF) to inhibit phosphatases. For immunoprecipitation experiments, 2 mg of whole-cell lysates were incubated for 1–2 hr at 4°C with the appropriated amount of mouse monoclonal anti-frataxin (Abcam) or mouse monoclonal anti-RNF126 (Santa Cruz) antibodies per sample. The immunocomplexes were then absorbed for 1–2 hr at 4°C on prewashed protein glutathione Sepharose beads (GE Healthcare) and finally washed three times in wash buffer (25 mM Tris-HCl [pH 7.5], 125 mM NaCl, 1% glycerol, 1 mM MgCl₂, 0.5% Igepal CA-630) supplemented with complete protease inhibitor cocktail, 5 mM NEM, 1 mM NaV, and 5 mM NaF. After washing, immunocomplexes were resuspended in 2× Laemmli sample buffer, boiled for 5 min, resolved by SDS-PAGE, and analyzed by WB with rabbit polyclonal anti-frataxin (Abcam) or mouse monoclonal anti-RNF126 (Santa Cruz). Densitometric analyzes were performed using ImageLab software.