Procedure for separating nuclear, membrane and cytoplasmic cell fractions using centrifugation methods.
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 cell suspension through a 27 gauge needle 10 times (or until all cells are lysed).
Leave on ice for 20 min.
Centrifuge sample at 720 xg (3,000 rpm) for 5 min. The pellet will contain nuclei and the supernatant will contain cytoplasm, membrane and mitochondria.
Transfer supernatant into a fresh tube and keep on ice. This will be dealt with in Steps 8–11.
Wash nuclear pellet from Step 4 with 500 μL 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 6 in TBS with 0.1% SDS. Sonicate the suspension briefly to shear genomic DNA and homogenize the lysate (3 s on ice at a power setting of 2-continuous).
Centrifuge the supernatant recovered in Step 5 at 8,000 rpm (10,000 x g) for 5 min. The pellet contains mitochondria. Transfer the supernatant into a fresh tube and keep on ice: this is the cytoplasm and membrane fraction.
Process the mitochondrial pellet from Step 8, 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 8 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 gauge 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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