Answer:
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 µg/ml aprotinin, 1 µg/ml leupeptin, 2 µg/ml antipain, 
10 µg/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, 4°C 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, 4°C X 10 min. 
9) Collect supernatant and combine with supernatant from first homogenization. 
10) Centrifuge pooled supernatants in Sorvall SS-34 rotor or equivalent at 40,000 X g, 4°C for 90 
min (or ultracentrifuge at 100,000 X g for one hour if available). 
11) Save pellets, these are the crude membrane fraction. Resuspend in 2.5 ml for every gram of 
brain used. 
12) Rehomogenize using hand held Dounce glass-glass homogenizer. Should yield ≈10-15 mg 
protein/ml. 
Can modify protocol to brains from any mammalian species by proportionally adjusting volumes. 
 
from the NeuroMab website