1. Wash cells with PBS.
2. Add 1 ml PBS to 54 cm2 dish. Scrape cells into the 1 ml.
3. Pipet cells into 15 ml orange cap tube. Spin in swinging bucket centrifuge in cold room for 15 minutes at 2000 rpm.
4. Pour off supernatant. Resuspend cells in at least 9 volumes wash buffer. (From pooled cells from 3 dishes, use 900 ml wash buffer at minimum.)
5. Homogenize cells in glass Dounce homogenizer with 25 strokes.
6. Put homogenate in a 10 ml Oak Ridge tube Add wash buffer to tube to bring volume up to close to the top of tube so the tube doesn’t collapse.
7. Spin in Sorvall Superspeed at 20,000 rpm (47,800 X g) for 10 minutes at 4øC to pellet membranes.
8. Wash 3 times with wash buffer. For each wash, pipet the pellet off the wall and spin again.
9. Resuspend in 0.32 M sucrose.
10. Quantitate protein with BCA kit.
Tissue culture number:
Cell resuspension volume:
Membrane resuspension final volume:
Date of BCA assay:
Wash buffer recipe (1 liter):
0.32 M sucrose 109.5 g (MW 342.3)
10 mM Tris-Cl pH 7.5 100 ml of 1 M Tris-HCl at pH 7.5
5 mM MgCl2 2 ml of 2.5 M MgCl2 stock
10 mM GTP add fresh each time: 5 ml of 100 mM stock into 50 ml wash
50 mM NaCl 10 ml of 5 M NaCl stock