on ice visible blood clots and the outer membrane of the meninges with their constituent blood vessels were removed the brains were sliced into cubes approximately 2 cm on an edge quick frozen in liquid n 2 and stored at 70 u00b0 c distilled water was used to make all solutions and ph values were adjusted versus standards at the temperature of use all steps were performed at 4 u00b0 c unless otherwise noted four kg of tissue about 12 whole adult bovine brains were thawed in 4 liters of 0 15 m nh 4 2 so 4 homogenized in a waring blender and adjusted to ph 4 5 using 6n hcl while vigorously mixing with a 6 inch diameter propeller stirrer after 1 hour the homogenate was centrifuged at 13 800 u00d7 g for 40 minutes the supernatant adjusted to ph 6 75 with 1 m naoh and 200 g liter of nh 4 2 so 4 1 52 m slowly added while stirring after centrifugation at 13 800 u00d7 g for 30 minutes 250 g of nh 4 2 so 4 was added per liter of supernatant 3 41 m the mixture was recentrifuged the resulting pellet dissolved in 200 ml of water dialyzed 18 hours in 6 000 8 000 m r cutoff bags spectrum medical industries los angeles calif versus two 14 liter volumes of water and lyophilized lyophilized protein from the salt precipitate of 16 kg of brain was.