Dynabead protein g beads

Lysates from C2C12 myotubes, soleus, and triceps were obtained by homogenization in co-IP buffer (10% NP-40, 20% 20 mM NaF, 1% Triton X-100) supplemented with complete protease inhibitor tablet (Roche Applied Science) and 1 mM leupeptin and 1 mM pepstatin A (MilliporeSigma). Cells were collected and lysed at 4°C for 30 minutes. After centrifugation (16,000g, 4°C, 20 minutes), the soluble fractions were collected, and the concentration was measured using a colorimetric BCA assay (23225; Thermo Fisher Scientific). Soluble homogenates were precleared with Dynabead Protein G beads (Thermo Fisher Scientific) for 1 hour, and supernatants were incubated with the specific antibodies directed against the protein of interest at 4°C for 12 to 24 hours. Dynabead Protein G beads were then added for 2 hours to capture the immune complex. Beads were washed 3 times with co-IP buffer supplemented with 0.1% CHAPS. For all experiments, 2 negative controls consisted of a sample lacking the primary antibody and a sample incubated with another primary antibody from the same serotype as the antibody of interest. Resulting beads were eluted with Laemmli buffer and subjected to SDS-PAGE followed by immunoblot.