All tags Cell Fractionation Kits Subcellular fractionation protocol

Subcellular fractionation protocol

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

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Reagents

Subcellular fractionation buffer


Stocks
250 mM Sucrose

-

4.28 g

20 mM HEPES (7.4)

1 M

1 mL

10 mM KCI

-

0.0373 g
1.5 mM MgCl2

1 M

75 μL
1 mM EDTA

0.5 M

100 μL
1 mM EGTA

0.5 M

100 μL

Just before use, add the following per 10 mL:


StocksPer 50 mL
1mM DTT

1M

  10 μL
PI Cocktail (III)

-

  50 μL


​Procedure

  1. All centrifugations should be done at 4°C.
  2. Lyse cells on 10 cm plates using 500 μL of buffer. Scrape plates immediately and place in 1.5mL Eppendorf tube.
  3. Pass lysate through a 25 gauge needle 10 times using a 1 mL syringe.
  4. Leave on ice for 20 min.
  5. Centrifuge out the nuclear pellet (P1) at 720 x g (3,000 rpm) for 5 min. Wash once by adding 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. Remove the buffer and resuspend the pellet in the nuclear buffer (standard lysis buffer with 10% glycerol and 0.1% SDS added). Sonicate the nuclear pellet briefly (3 sec) on ice at a power setting of 2-continuous.
  6. Place the supernatant from the first centrifuge step in a fresh labeled Eppendorf tube. Centrifuge at 8,000 rpm (10,000 x g) and remove the supernatant. This is the cytosolic and membrane fraction.
  7. If the mitochondrial fraction is desired, save the pellet from Step 5 and wash as with the nuclear pellet.
  8. For a membrane fraction, centrifuge the supernatant 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.
  9. Optional: Concentrate the supernatant by centrifuging through the filter unit. This concentrates the cytosol down to approximately 50–75 μL.



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