Protein g sepharose 4 fast flow beads

Crude lysates of testis and sperm from mouse or bull were incubated for 2 hr at 4°C with Protein G-Sepharose 4 Fast Flow beads (GE Healthcare) which were washed once with distilled water and twice with homogenization buffer (20 mM Tris-HCl pH 7.0, 1 mM EDTA, 1 mM EGTA, 10 mM Benzamidine, 1 mM PMSF, 0.1 mM of TPCK, and 0.1% 2-mercaptoethanol). It was then spun-down at 10,000 × g for 1 min and the supernatant was incubated with 5 μg of the appropriate antibody or diluted rabbit preimmune serum as a negative control, overnight with gentle rocking at 4°C. Following day, Protein G-Sepharose 4 Fast Flow beads (GE Healthcare) were washed once with distilled water and twice with 1× TTBS. Each extract/antibody solution was incubated with the beads by rocking for 2 hr at 4°C. After incubation, the beads were washed five times with 1× TTBS. After washing, the beads were resuspended in 2× SDS reducing sample buffer (6% SDS, 25 mM Tris-HCl pH 6.5, 50 mM dithiothreitol (DTT), 10% glycerol, and bromophenol blue), boiled for 10 min and centrifuged at 10,000 × g for 10 min. The supernatants were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by western blot analysis.