Nuclear Extract Protocol: 4 brains
Stock of 200 mM PMSF prepared with isopropanol, final concentration is 0,1 mM
On ice:
1. Cut brains into small pieces using scissors
2. Add ca. 16 ml Homogenization Buffer (Adam) (0,3 M Sucrose, 0,1 mM
EDTA, 10 mM HEPES, 10 mM KCl)
3. Dounce with 10 strockes, cool on ice and apply another 10 strockes
4. Filter into a small beaker through 2 layer of presoked gaze in Homogenization
Buffer, change gaze if necessary to get rid of cell debris
5. add Homogenization Buffer to 10 ml/brain (so total 20 ml for 4 brains)
6. Mix homogenate with 1 volumes of C (25 ml: 50% (5 vol=20,8 ml) of
OptiPrep, 1 vol=4,2 ml of - 150 mM KCl (1250 µl), 15 mM EDTA (750 µl),
10 mM HEPES pH 7.9 (250 µl))
7. add 10 ml of 29% solution of OptiPrep (25 ml: 7,25 ml OpriPrep, 0,25 M
Sucrose (2,14g), 25 mM KCl (208,3 µl), 5 mM MgCl2 (41,7 µl), 10 mM
HEPES (250 µl), 15 mM EDTA (750 µl)) to the bottom of the centrifuge tube
(do not touch the sides!!!)
8. Split the homogenate into the tubes (run it slowly down the side of the tube)
9. balance the tubes (within 0,05 g of each other)
10. centrifuge @ 7500 rpm for 30 min @ 1ºC (SW-32-TI)  take 1 ml sample
from the supernatant for Western analysis
11. Dry in cold room inverted for 10 min
12. Suspend the pellet in 10 ml Nuclear Lysis Buffer (re-suspend in 1-2 ml first,
transfer the solution into the homogenizer, don’t decant!!! wash the bottom of
the tube with the rest of the buffer)
13. apply 10-15 strokes with pestle A to homogenize the pellet and lyse the cells
14. Transfer the nuclei into a small beacon with a stirring bar and bring up the
volume to 10 ml (for 4 brains)
15. add drop wise 1/20 volume (500 µl) of saturated (NH4)2SO4 over 25 min, on
ice!
16. centrifuge @ 20000 rpm for 30 min @ 1 ºC (SS-34), keep supa
17. add 0,4 g/ml of finely powdered (NH4)2SO4 (4 g) over 15 min on ice!, let sit
for another 15 min
Robertson Lab
18. centrifuge for 30 min @ 24000 rpm @ 1ºC (ultracentrifuge)
19. Suspend the pellet in 200 µl of Dialysis Buffer
20. Dialyse the NE against 100 ml of Dialysis Buffer, in the cold room under
stirring
21. Change dialysis buffer after 2-4 h, repeat after another 4 h
22. centrifuge @ 1 ºC (Eppendorf) for 10 min
23. collect supernatant
24. Measure concentration (Bredford 2µl, 5 µl)
25. aliquote into 100 µl samples
26. store @ -80 ºC
Robertson Lab