Subcellular fractionation buffer
|Stocks||50ml 1 x solution|
|250 mM Sucrose|
|20 mM HEPES (7.4)|
|10 mM KCl|
| 0.0373 g|
|1.5 mM MgCl2|
| 75 μl|
|1 mM EDTA|
| 100 μl|
|1 mM EGTA|
| 100 μl|
At time of use: Take 10 ml and add the following:
|Stocks||50 ml 1 x solution|
| 10 μl|
|PI Cocktail (III)|
| 50 μl|
Note: All centrifugations should be done at 4°C.
- Lyse cells on 10 cm plates Using 500 μl of buffer. Scrape plates immediately and place in 1.5 ml eppendorf tube.
- Then pass lysate through a 25 G needle 10 times using a 1 ml syringe.
- Leave on ice for 20 min.
- Centrifuge out the nuclear pellet (P1) at 720 G (3000 rpm) for 5 min. The nuclear pellet should be washed once by adding 500 μl of fractionation buffer again. Disperse the pellet with a pipette and pass through a 25 G needle 10 times. Centrifuge again at 3000 rpm for 10 min. Remove the wash buffer, and resuspend the nuclear 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.
- Remove the supernatant and place in a fresh labeled eppendorf tube.
- Centrifuge the supernatant again at 8000 rpm (10,000 G). Remove the supernatant again. This is the cytosolic and membrane fraction.
- If the mitochondrial fraction is desired, save the pellet from step 6, wash as with the nuclear pellet and resuspend in the same buffer as above.
- For a membrane fraction: centrifuge the supernatant in an ultracentrifuge. Centrifuge at 40,000 rpm (100,000 G) for 1 hr. Wash this pellet by adding 400 μl of the fractionation buffer to the pellet. Resuspend by pipetting. Use a 25 G needle as above. Then 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 should concentrate the cytosol down to approximately 50 - 75 μl.
Dr. Richard Patten
Tufts-New England Medical Centre
Molecular Cardiology Research Centre