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Protein Extraction and Western Blotting Epididymis was harvested from anesthetized rats. Tissue was cut into small pieces and rinsed several times in PBS/protease inhibitors to remove most of the sperm. Tissue was homogenized in 10 ml/g of buffer containing 250 mM sucrose, 18 mM Tris, 1 mM EDTA, and complete protease inhibitor (Roche Applied Science, Indianapolis, IN), adjusted to pH 7.4 with HEPES, using a PRO 200 homogenizer followed by 20 strokes in a glass potter fitted with a Teflon pestle (Thomas Scientific, Swedesboro, NJ). The protein concentration was determined with the bicinchoninic acid protein assay (Pierce Biotechnology, Rockford, IL) using albumin as a standard. Protein extracts were diluted in Laemmli sample buffer, boiled for 1 min, and loaded onto Tris-glycine polyacrylamide 4%-20% gradient gels (PAGEr Duramide Precast Gels, 4%-20% Tris-glycine Gels; Cambrex, Rockland, ME). After SDS-PAGE separation, proteins were transferred onto an Immun-Blot polyvinylidene difluoride membrane (Bio-Rad Laboratories). Membranes were blocked in Tris-buffered saline (TBS) containing 5% nonfat dry milk and then incubated overnight at 4°C with the primary antibodies at a concentration of 0.5 1g/ml in TBS containing 2.5% milk.