A two-phase polymer method for isolation of maturing goat sperm plasma membrane

An aqueous two-phase polymer method originally developed for the isolation of plasma membrane from mature goat epididymal spermatozoa (Rana, A.P.S. and Majumder, G.C., Prep. Biochem., 17, 261, 1987) has been found to be unsuitable for the maturing spermatozoa derived from caput and corpus epididymides because of significant contamination of the isolated membrane with intact cells. A modified method has been developed by manipulating the centrifugal force (required for membrane sedimentation) for the isolation of maturing sperm plasma membrane of high yield (approximately 55%) and purity as judged by marker enzyme assays and phase contrast and electron microscopic analyses. The method consists of treatment of intact spermatozoa with 1.25 mM EDTA, dispersion of these cells to a two-phase polymer system comprising 5.5% 252-Kd dextran and 4.2% 20-Kd polyethylene glycol compound and subsequent centrifugation at 12,000 X g for 30 min when the two phases separate out and membranes sediment at the interphase. The repeatation of the two-phase fractionation step yielded greater purity of the plasma membrane.