Factors Influencing the Yield and Purity of Goat Sperm Plasma Membranes Isolated by Means of An Aqueous Two-Phase Polymer System

Abstract An improved aqueous two-phase polymer method has been developed for the isolation of sperm plasma membranes by manipulating various parameters that influence markedly the purity as well as yield of the membrane. The method consists of hypotonic shock of intact spermatozoa with 1.25 mM EDTA to dissociate the plasma membrane and dispersion of these cells to a two-phase polymer system consisting of 5.5% 252-Kd dextran and 4.2% 20-Kd polyethylene glycol prior to centrifugation at 9700 x g for 30 min when the two polymer phases are separated, the membrane fraction sediments at the interphase. The resulting membrane fraction was purified further by repeating the two-phase fractionation step. The yield of the membranes was approx. 35–40%, based on the recovery of the membrane-bound marker enzymes alkaline phosphatase and 5′-nucleotidase. The isolated membranes showed a high degree of purity as evidenced by phase contrast and electron microscopic studies and analyses of marker enzymes characteristic of c...