Dynabead m 280 sheep anti mouse igg

For each co-IP sample, one confluent 10 cm dish of cells was used. Cells were washed with PBS, scraped, and centrifuged at 200 g for 5 min to pellet. Cell pellets were snap frozen for storage at −80°C until use. Dynabead M-280 Sheep Anti-Mouse IgG (Invitrogen) magnetic beads were washed three times using TBS+0.05% Tween-20 (TBST) before incubating with 5 µg anti-Strep antibody (ref antibody list) for 45 min with rotation at room temperature. Cell pellets were resuspended and lysed in 500 µl of gentle, non-denaturing lysis buffer (20 mM Tris-HCl pH 8.0, 137 mM NaCl, 1% NP-40 (Igepal), 2 mM EDTA, Protease Inhibitor Cocktail Set III (EMD Millipore)) on ice for 30 min. After cell lysis, lysates were centrifuged at 20,000 g at 4°C for 10 min. Antibody bound beads were washed three times with TBST before resuspending in 100 µl of the gentle lysis buffer. The remaining of the cleared lysate was added to the resuspended beads and incubated overnight at 4°C with rotation. After overnight incubation, About 20 µl or 4% of the cleared lysate was set aside as the INPUT sample to check for antibody integrity and protein expression. The remaining IP samples were washed in chilled lysis buffer three times, before resuspending in 60 µl lysis buffer. INPUT and IP samples were carried forward to Western blotting.