10mm Tris, 2mm EDTA, pH 7.5
For 25 mL, the use of (a) 250uL 1M Tris + (b) 500uL of 0.1 M EDTA.
Cell Fractionation Protocol
sugar with 20mm Tris buffer, 0.5 M EDTA, and 0.1 M NaCl
To 2 mL, the use of (a) 40uL 1M Tris + (b) 10ul of 0.1 M EDTA + (c) 200ul 1M NaCl +
(D) 1.2g sucrose + (e) water 1.75mL
Also make sugar without sugar 20ml buffer (to thin the membrane fraction)
(A) 400uL 1M Tris + (b) 100uL 0.1 M EDTA + (c) 2 mL of 1 M NaCl + (d) water 17.5mL
Make 1mL on without EDTA buffer (for resupension during the last step)
(A) 1 M Tris 20uL, 100uL 1M NaCl
1 mM DTT
First, make a 0.1 M DTT (15.4mg DTT in 1mL of water), then take 100uL it, and add 10 ml of 10mm Tris, 2mm EDTA (= 100uL 1M Tris, 200ul of 0.1 M EDTA)
NEM 40mm in 20mm Tris
To 5 mL, 25mg weight NEM + 100uL of 1M Tris
Cell Fractionation Protocol
protease inhibitors (pefabloc)
made on 400mm, or 9.58mg in 100uL water
use at 1% final concentration
if using a complete inhibitor (Roche)
1 tablet dissolved in water 2 mL
aliquot and freeze
used at a final concentration of 4% (ie 4UL per 100uL sample)
1. Aspiration media
2. wash 1x with 10 mL of PBS to each plate
3. Add 1 mL of PBS, and the cells friction with cell scraper to eppendorf tubes; add more PBS and scrape again if they have more cells. Wash scraper between the various types of samples.
4. Centrifuge to pellet the cells for 1 minute.
(A) Lyse in 10mm Tris, 2mm EDTA, 1 mM DTT
(B) Wash 2x with the above solution, then resuspend in 200ul solution of
(C) Add 1: 1 with NEM 40mm, 20mm Tris and incubate on ice for 20 minutes.
5. Wash 2x with 10mm Tris, 2mm EDTA
6. resuspend in 400uL above buffer + 4UL PI
7. Keep on ice for 1 hour; whirlpool and unifies every 15 minutes
(During this time, centrifuge tubes and holders place on ice; turn on Beckman centrifuge and set it at 38,000 x g for 30 min, 4 ° C)
8. Put the needle through 26.75g 6x (be careful not to get too many bubbles)
9. Points 500uL 60% sugar, then place the sample above into the centrifuge tube
Also obtain the cytosolic fraction, 10% sucrose solution put on top of 60% sucrose before applying the sample; after spinning, the supernatant fraction will cytosol above
10. The tube Balance
11. Spin at 38,000 x g, 30 min. in a swing-out rotor
12. Take out the membrane fraction among the fractions above (s) and a layer of 60% sucrose (approximately Remove 300-400uL). Be careful to not take sucrose.
13. Remove excess liquid and resuspend the pellet into an appropriate buffer. These pellets will be a nuclear fraction.
14. Dilute membrane fraction of at least 6x in sucrose buffer without sucrose.
15. spin at 100,000 x g for 15 minutes
16. resuspend any of the required volume of 20mm Tris, 0.1 M NaCl + 1% of 400mm pefabloc
17. To save the membrane fraction of membrane proteins, it is possible to + 5% glycerol and put it in the freezer -80, but when taking it out again, one must resuspend the membrane in washing buffer (buffer ie sugar without sugar), and then spin membrane down again at 100,000 xg