Horseradish peroxidase conjugated anti rabbit igg

Total protein was extracted and then separated with a 10% (w/w) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, and the resulted proteins from the gel were blotted onto polyvinylidene difluoride (PVDF) membranes. The blot was blocked with 5% (w/w) skimmed milk for 2 h at room temperature and subsequently incubated overnight with the primary antibody at 4°C. Afterward, the blot was incubated with the antibody of horseradish peroxidase-conjugated anti-rabbit IgG (CWBio, Beijing, China) for 2 h. The immunoreaction was detected using the ECL Western Blot Kit (CW00495, CWBio, Beijing, China), and the ChemiDoc™ XRS imaging system (Bio-Rad Laboratories Inc., Hercules, CA, United States) was used to record the chemiluminescence on blots. The primary CBF1 antibody was ordered from GenScript Company (Nanjing, China).

Total sperm protein was isolated, and protein concentration was determined using the bicinchoninic acid method. About 15 μg protein was electrophoresed on the 8% sodium dodecyl sulfate–polyacrylamide gel for 90 min and transferred onto polyvinylidene difluoride membranes (GE Healthcare, Pittsburgh, PA, USA). The protein was incubated with anti-NMDAR2A/NMDAR2B (Abcam, Cambridge, UK; 1:1000) as the primary antibodies and horseradish peroxidase-conjugated anti-rabbit IgG (CWBIO, Beijing, China; 1:5000) as the secondary antibody at room temperature for 60 min. The membranes were then visualized using an enhanced chemiluminescence detection kit (Proteintech, Wuhan, China) under a Bio-Rad gel imaging system (Bio-Rad, Hercules, CA, USA). GAPDH was blotted as the negative control. The experiments were repeated at least three times.