Biochemical Techniques and Instrumentation

Procedure

Note: All centrifugations should be done at 4°C.

1. Lyse the cells on 10 cm plates using 500 μl of buffer. Scrape the cells from plates immediately and place in 1.5 ml eppendorf tube
2. Then pass lysate through a 25 G needle 10 times using a 1 ml syringe.
3. Leave on ice for 20 min.
4. Centrifuge out the nuclear pellet (P1) at 720 G (3000 rpm) for 5 min.
5. Wash the nuclear pellet once again by adding 500 μl of fractionation buffer again.
6. Disperse the pellet with a pipette and pass through a 25 G needle 10 times.
7. Centrifuge again at 3000 rpm for 10 min.
8. Remove the wash buffer, and resuspend the nuclear pellet in the nuclear buffer (standard lysis buffer with 10% glycerol and 0.1% SDS).
9. Sonicate the nuclear pellet briefly for 3 sec on ice.
10. Remove the supernatant and place in a fresh labeled eppendorf tube.
11. Centrifuge the supernatant again at 8000 rpm for 20 min.
12. Remove the supernatant again. This is the cytosolic and membrane fraction.
13. If the mitochondrial fraction is desired, save the pellet from step 6, wash as with the nuclear pellet and resuspend in the same buffer as above.
14. For a membrane fraction, Centrifuge the supernatant in an ultracentrifuge.
15. Centrifuge at 40,000 rpm for 1h.
16. Wash this pellet by adding 400 μl of the fractionation buffer to the pellet.
17. Resuspend by pipetting. Use a 25 G needle as above.
18. Then re-centrifuge for 45 min.
19. Resuspend the membrane pellet in the same buffer as used for the nuclei.
20. Optional : Concentrate the supernatant by centrifuging through the filter unit. This should concentrate the cytosol down to approximately 50 - 75 μl.