Goat anti rabbit a10533 horseradish peroxidase conjugated antibodies

For protein level analyses, proteins were extracted from germ cells using RIPA buffer (Santa Cruz) containing 1× protease inhibitor cocktail (Roche). Protein concentration was calculated using a Bicinchoninic acid (BCA) protein assay kit (Pierce). Lanes of 6%, 10%, 15%, and 4–15% gradient SDS polyacrylamide gels (Bio-Rad) were loaded with 20 µl of 1 mg/ml protein extract. Following protein separation via standard SDS–PAGE, proteins were transferred to polyvinylidene difluoride (PVDF) membranes using the Trans-Blot Turbo Western transfer system (Bio-Rad). Primary antibodies and dilution used are presented in Supplemental Table S2. At a 1:5000 dilution, goat anti-mouse (62-6520) and goat anti-rabbit (A10533) horseradish peroxidase–conjugated antibodies (Invitrogen) were used as secondary antibodies. The presence of antibodies on the PVDF membranes was detected via treatment with Pierce ECL Western blotting substrate (Thermo Scientific) and captured using the Syngene XR5 gel documentation system. Protein levels were assessed using Image J (NIH).

For protein level analyses, proteins were extracted from germ cells using RIPA buffer (Santa Cruz) containing 1× protease inhibitor cocktail (Roche). Protein concentration was calculated using a BCA protein assay kit (Pierce). Lanes of 4–15% gradient SDS polyacrylamide gels (Bio-Rad) were loaded with 20 µl of 1 mg/ml protein extract. For STA-PUT, 20 µl of 0.1 mg/ml protein extracts from purified leptotene/zygotene and pachytene/diplotene stage spermatocytes and round spermatids were loaded per lane on SDS polyacrylamide gels. Following protein separation via standard SDS–PAGE, proteins were transferred to polyvinylidene difluoride (PVDF) membranes using the Trans-Blot Turbo Western transfer system (Bio-Rad). Primary antibodies and dilution used are presented in Supplemental Table S2. At a 1:5000 dilution, goat anti-mouse (62-6520) and goat anti-rabbit (A10533) horseradish peroxidase–conjugated antibodies (Invitrogen) were used as secondary antibodies. The presence of antibodies on the PVDF membranes was detected via treatment with Pierce ECL Western blotting substrate (Thermo Scientific) and captured using the Syngene XR5 gel documentation system. Protein levels were assessed using Image J (NIH).