My current protocol is: 

Procedure:

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

1) Scrape cells on 10 cm plates using 5 ml of cold PBS plates immediately and place in 15 ml falcon tube. 2) Pellet cell at 600 rpm 4°C for 3 min, then transfer to a 1.5 ml eppendorf.

3) Resuspend in 500ul of the fractionation buffer (cytosolic, membrane, nucleus and mitochondria).

4) Then pass lysate through a 25 G needle 10 times using a 1 ml syringe. This disrupts the membranes but not nucleus.

5) Leave on ice for 20 min.

6) Centrifuge out the nuclear pellet (P1) at 720 G (3000 rpm) for 5 min. The supernatant here contains the cytosolic proteins and should be placed in a freshly labeled eppendorf tube. Can clear the supernatant here by spinning at 14000rpm for 15min.

7) The nuclear pellet should be washed once by adding 500 μl of fractionation buffer again. Disperse the pellet with a pipette and pass through a 25 G needle 10 times.

8) Centrifuge again at 3000 rpm for 10 min. Remove the wash buffer, and resuspend the nuclear pellet in the nuclear buffer (RIPA: standard lysis buffer with 10% glycerol added). Sonicate the nuclear pellet briefly (3 sec) on ice at a power setting of 2-continuous. Can clear the supernatant here again of big membrane proteins using 14000rpm spin for 10min at 4degC.

The fractionation buffer is sucrose, HEPES, KCL, MgCL, EDTA and EGTA with DTT.