Hybri slot manifold system

Human spermatozoa lysates were prepared from the resulting pellets. Pellets were resuspended in RIPA buffer and incubated for 15 min at 4 °C. The samples were then centrifuged (14,000× g for 20 min) and total protein concentration was quantified using a BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.
The phosphorylation state of spermatozoa proteins was used as a biomarker to evaluate sperm capacitation [26 (link)]. Protein samples were diluted to a final concentration of 50 ng/µL and transferred to a PVDF membrane using a slot blot technique in a Hybri-slot manifold system (Biometra, Göttingen, Germany). A Ponceau S staining solution (MB19201, NZYTech, Lisboa, Portugal) was used for total protein normalization. The resulting membranes (n = 6 for each condition) were incubated overnight at 4 °C with a mouse anti-phosphotyrosine monoclonal antibody (1:1000, Clone 4G10, #05-321, Merck Millipore, Temecula, CA, USA). Membranes were then incubated with a goat anti-mouse IgG antibody (1:5000, AP308P, Sigma-Aldrich, St. Louis, MO, USA). Blots were visualized with Clarity™ Western ECL Substrate (Bio-Rad, Hercules, CA, USA) and read using a Bio-Rad ChemiDoc XR system (Bio-Rad, Hercules, CA, USA). Densities from each band were quantified using Image Lab Software (Bio-Rad, Hercules, CA, USA).