All centrifugations should be done at 4°C. Samples should be kept on ice throughout the procedure.
Transfer cells from 10 cm plates into 500 μL fractionation buffer, eg by scraping. Incubate 15 min on ice.
Using 1 mL syringe pass cells suspension through a 27 gauge needle 10 times (or until all cells are lysed).
Leave on ice for 20 min.
Centrifuge sample at 720 x g (3,000 rpm) for 5 min. Pellet contains nuclei. Supernatant contains cytoplasm, membrane and mitochondria.
Transfer supernatant into a fresh tube and keep on ice. This will be dealt with in steps 9-13.
Wash nuclear pellet remained after step 6 with 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. Discard the supernatant and keep the pellet that contains nuclei.
Resuspend the pellet from step#7 in TBS with 0.1% SDS. Sonicate the suspension briefly to shear genomic DNA and homogenise the lysatae (3 sec on ice at a power setting of 2-continuous).
Continue with cytoplasm, membrane and mitochondria fraction from step 6.
Centrifuge supernatant recovered in step #6 at 8,000 rpm (10,000 x g) for 5 min. Pellet contains mitochondria. Supernatant contains cytoplasm and membrane.
Transfer supernatant from step 10 into a fresh tube and keep on ice. This is the cytoplasm and membrane fraction.
Process the mitochondia pellet from step 11, as described for the nuclear pellet in step 7, to obtain mitochondrial lysate in TBS/0.1% SDS.
For a membrane fraction, centrifuge the supernatant from step 11 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.
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