All centrifugation 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 (recipe below), 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 xg (3,000 rpm) for 5 min. The pellet will contain nuclei and the supernatant will contain cytoplasm, membrane and mitochondria.
Wash nuclear pellet remaining 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 in TBS with 0.1% SDS. Sonicate the suspension briefly to shear genomic DNA and homogenize the lysate (3 sec on ice at a power setting of 2-continuous).
Nuclear extraction and fractionation buffer
Add per L
HEPES (pH 7.4)
Just before use, add the following per 10 mL:
Per 50 mL
1 mM DTT
PI Cocktail (III)
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