Secreted Epididymal Glycoprotein: MATERIALS AND METHODS(7)
Gels were stained with Coomassie blue R250 using standard procedures. Western blots were prepared from parallel gels, blocked with 4% (w/v) nonfat dry milk (‘‘Marvel’’) in PBS for at least 3 h at room temperature, and probed with McAb supernatants or polyclonal antisera (diluted 1:1000) for 2 h. After washing three times in PBS, blots were probed with the appropriate Px-RAM or Px-SAM (diluted 1:5000) for 2 h. Bound antibody was detected on x-ray film by chemiluminescence procedures (NEN Life Sciences, Boston, MA). read
Triton X-114 Partitioning, PI-PLC Digestion, and Effects of Dissociating Reagents on Binding of 2D6 Antigen to the Plasma Membrane of Cauda Spermatozoa
To determine the strength of association between the cauda form of 2D6 antigen and the plasma membrane, spermatozoa were exposed to Triton X-114, PI-PLC, and different dissociating reagents followed by an analysis of its partitioning between the insoluble (membrane bound) and soluble fractions. For this purpose, washed cauda sperm pellets were resuspended in 1% (v/v) Triton X-114 in PBS/1 mM Pefabloc, incubated at 4°C for 30 min, and centrifuged at 1400 X g for 10 min. The supernatant was removed and overlaid on a 10% (w/v) sucrose cushion containing 0.06% Triton X-114 at 4°C. Samples were heated to 37°C for 20 min to induce phase separation and centrifuged at 750 X g for 10 min at room temperature. Aqueous and detergent phases were removed for SDS-PAGE analysis and Western blotting. 2B1 glycoprotein, which partitions strongly into the detergent phase, was used as an internal control.