What is the protocol for preparing mammalian brain fractions?
1) Weigh two adult rat brains, should be ≈ 4-5 grams total. 2) Place in 40 ml of ice-cold homogenization buffer (0.32 M sucrose, 5 mM Na Phosphate buffer pH 7.4, 0.1 M Na fluoride, with fresh addition of 2 g/ml aprotinin, 1 g/ml leupeptin, 2 g/ml antipain, 10 g/ml benzamidine and 0.5 mM PMSF) in a 50 ml Potter-Elvehjem Tissue Grinders 3) Homogenize on ice using 10 strokes of a Teflon-glass homogenizer attached to a 3/8 variable speed drill at 2,500 rpm. 4) Centrifuge homogenate in Sorvall SS-34 rotor or equivalent at 750 X g, 4C X 10 min. 5) Collect the supernatant and save (this has brain membranes = crude synaptosomes) 6) Recover additional membranes from pellet by scraping off top, lightly colored layer (do not disturb deeper layer of red blood cells). Save pellet as low speed pellet or LSP. 7) Rehomogenize pellet using 40 ml of buffer, this time use only 8 strokes. 8) Centrifuge this homogenate in Sorvall SS-34 rotor or equivalent at 750 X g, 4C X 10 min. 9) Collect supernatant and
