Verification of PGP9.5 Cross-Reactivity in Donkey Testes

Western blotting was performed to verify the cross-reactivity of rabbit anti-human PGP9.5 (7863-2004, Bio-Rad, Hercules, CA, USA) to the PGP9.5 present in donkey testes according to the previously reported protocol with minor modifications [2 (link),11 (link)]. The testicular samples, which had been stored at −80 °C, were thawed at room temperature and homogenized using a Polytron PT 1200 CL homogenizer (Kinematica AG, Littau, Lucerne, Switzerland). Sample preparation buffer (0.5 M Tris-HCL (pH 6.8), 0.1% glycerol, 10% sodium dodecyl sulfate (SDS), 0.05% 2-β-mercaptoethanol, and bromophenol blue in distilled water) was used to dilute the concentration of quantified protein to 2 mg/mL. After being heated in a boiling water bath for 15 min, 15-μL samples were loaded onto a 10% SDS-polyacrylamide gel and separated using a Mini-Protean system (Bio-Rad, Hercules, CA, USA). The samples were electrotransferred to a polyvinylidene difluoride membrane (Millipore) and blocked with skim milk (BD Biosciences, San Jose, CA, USA). The membrane was incubated with PGP9.5 antibody diluted to 1:500 in skim milk overnight at 4 °C. For the negative control, the membrane was treated with normal mouse immunoglobulin G (IgG) at the same concentration of primary antibody. Horseradish peroxidase-conjugated anti-mouse IgG diluted in skim milk (1:10,000) was used as a secondary antibody.