Cell Fractionation Protocol
Biochemistry, Molecular Biology, and Cell Biology Protocols >> Cell Fractionation Protocol
Solutions to be made fresh that day:
10mM Tris, 2mM EDTA, pH 7.5
For 25mL, use (a) 250uL 1M Tris + (b) 500uL 0.1M EDTA
Sugar buffer with 20mM Tris, 0.5M EDTA, and 0.1M NaCl
For 2mL, use (a) 40uL 1M Tris + (b) 10uL 0.1M EDTA + (c) 200uL 1M NaCl +
(d) 1.2g sucrose + (e) 1.75mL water
Also make up 20mL of sugar buffer without sugar (for diluting the membrane fraction)
(a) 400uL 1M Tris + (b) 100uL 0.1M EDTA + (c) 2mL 1M NaCl + (d) 17.5mL water
Make 1mL of above buffer without EDTA (for resupension during last step)
(a) 20uL 1M Tris, 100uL 1M NaCl
First, make 0.1M DTT (15.4mg DTT in 1mL water), then take 100uL of that, and add to 10mL of 10mM Tris, 2mM EDTA (=100uL 1M Tris, 200uL 0.1M EDTA)
40mM NEM in 20mM Tris
For 5mL, weigh 25mg NEM + 100uL 1M Tris
Protease inhibitors (pefabloc)
made at 400mM, or 9.58mg in 100uL water
use at 1% final concentration
if using complete inhibitors (Roche)
dissolve 1 tablet in 2mL water
aliquot and freeze
use at 4% final concentration (i.e. 4uL per 100uL of sample)
1. Aspirate the media
2. wash 1x with 10mL PBS for each plate
3. Add 1 mL PBS, and scrape cells with a cell scraper onto eppendorf tubes; add more PBS and scrape again if still have more cells. Wash scraper between different types of samples.
4. Centrifuge to pellet cells for 1 min.
(a) Lyse in 10mM Tris, 2mM EDTA, 1mM DTT
(b) Wash 2x with above solution, then resuspend in 200uL of above solution
(c) Add 1:1 with 40mM NEM, 20mM Tris and incubate on ice for 20min.
5. Wash 2x with 10mM Tris, 2mM EDTA
6. Resuspend with 400uL of above buffer + 4uL protease inhibitors
7. Keep on ice for 1 hour; vortex and homogenize every 15 min
(During this time, place centrifuge tubes and holders on ice; turn on Beckman centrifuge & set it at 38,000 x g for 30 minutes, 4 degrees Celsius)
8. Put through a 26.75g needle 6x (be careful not to get too many bubbles)
9. Place 500uL 60% sugar, then place sample on top into the centrifuge tubes
To also obtain the cytosolic fraction, place a 10% sucrose solution on top of the 60% sucrose before applying the sample; after spinning, the cytosolic fraction will be the supernatant on top
10. Balance the tubes
11. Spin at 38,000 x g, 30 min. in a swing-out rotor
12. Take out the membrane fraction in between the top fraction(s) and the 60% sucrose layer (Take out approximately 300-400uL). Careful not to take out sucrose.
13. Remove the rest of the liquid and resuspend the pellet into a suitable buffer. This pellet will be the nuclear fraction.
14. Dilute the membrane fraction at least 6x in the sucrose buffer without sucrose.
15. Spin at 100,000 x g for 15 minutes
16. Resuspend in whatever volume required of 20mM Tris, 0.1M NaCl with + 1% of 400mM pefabloc
17. To store membrane the membrane fraction of proteins, it is possible to +5% glycerol and put it into the -80 freezer, but when taking it out again, one should resuspend the membranes in wash buffer (i.e. the sugar buffer without sugar), and then spin down the membranes again at 100,000 x g