Subcellular fractionation protocol

Procedure for separating nuclear, membrane and cytoplasmic cell fractions using centrifugation methods.

Procedure

All centrifugations should be done at 4°C. Samples should be kept on ice throughout the procedure.

  1. Transfer cells from 10 cm plates into 500 μL fractionation buffer, eg by scraping. Incubate 15 min on ice. 
  2. Using 1 mL syringe pass cell suspension through a 27 gauge needle 10 times (or until all cells are lysed).
  3. Leave on ice for 20 min.
  4. 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.
  5. Transfer supernatant into a fresh tube and keep on ice. This will be dealt with in Steps 8–11.
  6. 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.
  7. 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).
  8. 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.
  9. 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.
  10. 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.
  11. Optional: concentrate the supernatant by centrifuging through the filter unit. This concentrates the cytosol down to approximately 50–75 μL.


Subcellular fractionation buffer
​


MW

mM

Add per L

Unit

HEPES (pH 7.4)

238.30

20

4.77 g

g

KCI

74.55

10

0.75 g

g

MgCl2

95.21

2

0.19 g

g

EDTA

292.24

1

0.29 g

g

EGTA

380.35

1

0.38 g

g


​Just before use, add the following per 10 mL:


StocksPer 10 mL
1mM DTT

1M

  10 μL
PI Cocktail (III)

-

  50 μL


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