Anti insulin receptor antibody

Cells were lysed with modified Ripa buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mM NaF, 1 mM Sodium Vanadate, 10 nM Calyculin A, Protease Inhibitors). Lysates were centrifuged at 15,000 g for 20 min at 4°C, and protein concentration was determined by Pierce BCA Protein Assay Kit (Thermo Scientific). 100μg of protein was diluted in a total of 200μl of modified Ripa buffer then precleared with protein A agarose beads (Life Technologies) at 4°C for 1 hr. The supernatants were then incubated with 1 μg of anti-insulin receptor antibody (Cell Signaling) at 4°C overnight. Twenty microliters of protein A agarose beads were then added/ml of lysate, and incubated at 4°C for 1 hr. The agarose beads (with the antibody-protein complex) were then collected by centrifugation (10 min, 14,000 g, 4°C). Supernatants were discarded and beads were washed 3 × with PBS. Finally, the beads were re-suspended in 20 μl of 2 × sample buffer, boiled for 5 min, electrophoresed on 10% Tris-glycine gels (Novex).