Secreted Epididymal Glycoprotein: MATERIALS AND METHODS(8)
To investigate the possibility that the form of the 2D6 antigen on cauda spermatozoa had a GPI anchor, washed sperm suspensions (100 |xl) were incubated with 5 U/ml of phosphatidylinositol-specific phospholipase C (PI-PLC) for 2 h at 37°C. Samples were centrifuged and SDS extracts prepared of the supernatant and pellets for SDS-PAGE and Western blotting. CD52 glycoprotein, which contains a GPI anchor, was used as an internal control.
The effects of dissociating reagents on partitioning of cauda sperm 2D6 antigen between soluble and membrane-bound fractions was investigated by resuspending washed sperm pellets in (A) 1 M KCl, pH 7.2; (B) 4 M urea adjusted to pH 7.2; (C) PBS adjusted to pH 3.0 with HCl; (d) PBS adjusted to pH 10.0 with NaOH; (E) PBS, pH 7.2, containing 0.4% (v/v) p-mercaptoethanol; (F) PBS containing 0.1 M EDTA pH 7.2; and (G) PBS, pH 7.2, containing 1% (v/v) NP40. Sperm suspensions were incubated at room temperature for 30 min and centrifuged at 10 000 X g for 20 min. Supernatants were removed and pellets reextracted with 1% SDS in PBS for 5 min at room temperature. Supernatants and pellet extracts were analyzed by SDS-PAGE/Western blotting and probed with 2D6, 2B1, and PBP antibodies. buy ventolin inhaler