Materials
Subcellular fractionation buffer
MW | mM | Add per L | |
HEPES (pH 7.4) | 238.30 | 20 | 4.77 g |
KCI | 74.55 | 10 | 0.75 g |
MgCl2 | 95.21 | 2 | 0.19 g |
EDTA | 292.24 | 1 | 0.29 g |
EGTA | 380.35 | 1 | 0.38 g |
Just before use, add the following per 10 mL:
Stocks | Per 10 mL | |
1mM DTT | 1M | 10 μL |
PI Cocktail (III) | - | 50 μL |
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 x g for 5 minutes. The pellet will contain nuclei and the supernatant will contain the 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 720 x g for 10 minutes. Discard the supernatant and keep the pellet that contains the 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 10,000 x g for 5 minutes.
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 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
Concentrate the supernatant by centrifuging through the filter unit. This concentrates the cytosol down to approximately 50–75 μL.