Lyse cells on 10 cm plates using 500 μL of buffer. Scrape plates immediately and place in 1.5mL Eppendorf tube.
Pass lysate through a 25 gauge needle 10 times using a 1 mL syringe.
Leave on ice for 20 min.
Centrifuge out the nuclear pellet (P1) at 720 x g (3,000 rpm) for 5 min. Wash once by adding 500 μL of fractionation buffer. Disperse the pellet with a pipette and pass through a 25 gauge needle 10 times. Centrifuge again at 3,000 rpm for 10 min. Remove the buffer and resuspend the pellet in the nuclear buffer (standard lysis buffer with 10% glycerol and 0.1% SDS added). Sonicate the nuclear pellet briefly (3 sec) on ice at a power setting of 2-continuous.
Place the supernatant from the first centrifuge step in a fresh labeled Eppendorf tube. Centrifuge at 8,000 rpm (10,000 x g) and remove the supernatant. This is the cytosolic and membrane fraction.
If the mitochondrial fraction is desired, save the pellet from Step 5 and wash as with the nuclear pellet.
For a membrane fraction, centrifuge the supernatant in an ultracentrifuge at 40,000 rpm (100,000 x g) for 1 h. Wash pellet by adding 400 μL of fractionation buffer. Resuspend by pipetting and pass through a 25 G needle. Re-centrifuge for 45 min. Resuspend the membrane pellet in the same buffer as used for the nuclei.
Optional: Concentrate the supernatant by centrifuging through the filter unit. This concentrates the cytosol down to approximately 50–75 μL.