Nuclear Extract Protocol:
Buffers for 6 livers:
90 ml Homogenization Buffer
Protease Inh. None
DTT
90 µl (1 mM)
PMSF
450 µl (200 mM)
195 ml Cushion Buffer
Protease Inh. None
DTT
195 µl
PMSF
975 µl
60 ml Nuclear Lysis Buffer
Protease Inh. None
DTT
60 µl
PMSF
300 µl
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 6 ml Homogenization Buffer (8ml/liver! Don’t forget to add DTT and
PMSF to the buffer!)
3. Dounce with 10 strockes, cool on ice and apply another 10 strockes
4. Filter in 50 ml tube through 2 layer of presoked gaze in HomoBuffer, change
gaze if necessary to get rid of cell debris
5. add HomoBuffer to 8 ml/liver (so total 48 ml for 6 livers)
6. Mix homogenate with 2 volumes of Cushion Buffer (96 ml)
7. add 10 ml of Cushion Buffer 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 @ 24000 rpm for 1 h @ 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/liver (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
Robertson Lab
14. Transfer the nuclei into a small beacon with a stirring bar and bring up the
volume to 60 ml (for 6 livers)
15. add drop wise 1/20 volume (3 ml) 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 over 25 min on ice!
18. centrifuge for 1 h @ 24000 rpm @ 1ºC (ultracentrifuge)
19. Suspend the pellet in 3 ml of Dialysis Buffer
20. Dialyse 3 ml of 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
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
250 ml Homogenization Buffer
0,3 M
Sucrose
25,67 g
10 mM
Hepes pH 7,9 2,5 ml of 1 M
10 mM
KCl
833 µl of 3 M
0,1 mM EDTA
50 µl of 0,5 M
2,2 M
10 mM
10 mM
0,1 mM
300 ml Cushion Buffer
Sucrose
225,72 g
Hepes pH 7,9 3 ml of 1 M
KCl
1 ml of 3 M
EDTA
60 µl of 0,5 M
Robertson Lab
Robertson Lab