Porcine brains, which were obtained within 30 min after death of the animals from a local slaughterhouse, were used to prepare receptor-rich membranes. The brains were immediately frozen in liquid N2; 50 g brain per 200 mL ice-cold buffer (0.32 M sucrose, 10 mM potassium phosphate buffer, pH 7.0, 1 mM EDTA) were homogenized twice for 15 sec in a blender and then for 1 min with an ultraturrax. The homogenate was centrifuged three times for 15 min at 1.400 g and 4°C to separate cellular debris. The supernatant was spun down at 100.000 g for 60 min. The resulting pellet was resuspended in buffer (as above but without sucrose). Aliquots were stored frozen at -80°C. Protein content was determined by the Lowry method, using bovine serum albumin as a standard.32-35
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