Nuclear Extract Protocol:
Organs used: 4x hearts, 6x spleen, 12x single testes, 5x lung
Stock of 200 mM PMSF prepared with isopropanol, final concentration is 0,1 mM
On ice:
1. Cut livers into small pieces using scissors
2. Add ca. 5 ml Homogenization Medium (D) to the dounce homogeniser
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 D, change gaze
if necessary to get rid of cell debris
5. add D to 15 ml total volume
6. Mix homogenate with 1 volumes of C (15 ml)
7. add 10 ml of E (30% solution) 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
15. add drop wise 1/20 volume (500 µl) of saturated (NH4)2SO4 over 25 min, on
ice!
16. centrifuge @ 13000 rpm for 30 min @ 1 ºC (SS-34), keep supa
17. add 0,4 g/ml of finely powdered (NH4)2SO4 over 25 min on ice!
18. centrifuge for 30 min @ 24000 rpm @ 1ºC (ultracentrifuge)
19. Suspend the pellet in 250 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
Robertson Lab
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
Buffers used:
20 mM
0,2 mM
20%
100 mM
1 L Dialysis Buffer
Hepes pH 7,9 20 ml of 1 M
EDTA
0,4 ml of 0,5 M
Glycerol
200 ml of 100%
KCl
7,455 g
100 ml Nuclear Lysis Buffer
10 mM
Hepes pH 7,9 1 ml
10%
Glycerol
10 ml
100 mM KCl
0,7455 g
3 mM
MgCl2
100 µl
0,1 mM
EDTA
20 µl
300 ml Homogenization Medium (D)
0,25 M
Sucrose
25,67 g
20 mM
Hepes pH 7,9 6 ml
25 mM
KCl
0,56 g
5 mM
MgCl2
0,5 ml
30 mM
120 mM
150 mM
300 ml Diluent (B)
MgCl2
3 ml
Hepes pH 7,9 36 ml of 1 M
KCl
3,36 g
A OptiPrep
C Working solution: mix 5 volumes of solution A (OptiPrep) with 1 volume of
solution B
E Gradient solution 30%: mix 6 volumes of solution C with 4 volumes of solution D
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