2.7 Co-immunoprecipitation
Initially, the extracted proteins from human testes were incubated with 7 µl of target antibodies overnight at 4 °C. Subsequently, the mixture of each sample was added to a microcentrifuge tube containing 40 μL of prewashed Protein A/G magnetic beads (88803, Invitrogen) and incubated for 2 h at room temperature with constant rotation. After washing with 1 × PBS three times, the coimmunoprecipitated proteins were eluted with standard 5 × SDS sample buffer and heated for 10 min at 100°C. Finally, the co-immunoprecipitants were separated on 10% SDS-polyacrylamide gels and PVDF membranes for immunoblot analysis, as described above.