1.
Collect cells
from culture. Suck off
media, wash with PBS, and suck off PBS.
Add 1 ml refrigerated PBS to 60 mm plates and scrape the cells into
bottom of tilted plate. Pipet into
1.5 ml tube and place on ice.
2.
Pellet cells
at 5000 rpm in microfuge for 10 minutes.
3.
Remove
supernatant and discard. Measure
the packed cell volume (pcv).
For 3 60 mm plates, it is about 25 microliters.
4.
Rapidly
resuspend the cell pellets in a volume of hypotonic buffer about 5 times the pcv. Centrifuge 5 minutes at 5000 rpm in
microfuge. This step removes salt from cells so
effective swelling can occur in next step, but if you do this step too slowly
proteins can leak out of the cells.
5. Discard the supernatant and resuspend packed cells in
hypotonic buffer to a final volume of 3 times the original pcv (step 3) and
allow to swell 10 minutes on ice.
6.
Transfer cells
to a glass Dounce homogenizer.
Homogenize with ten up-and-down strokes using a type B pestle. You can look at an aliquot of cells
that have been stained with Trypan blue.
The nucleus of lysed cells should be blue, and 80% to 90% of the cells
should be lysed.
7.
Transfer cells
back to fresh microfuge tubes.
Collect the nuclei by centrifuging 15 minutes at 6500 rpm in microfuge. Supernatant consists of cytoplasm and
can be saved for cytoplasmic extract if desired.
8.
Measure the
packed nuclear volume (pnv).
Resuspend the nuclei with a volume of low-salt buffer equal to « pnv. (pnv from 3 60 mm plates was about 50
microliters)
9.
Add a volume
of high-salt buffer equal to « pnv (from step 8). This should either be added dropwise
with mixing, or, if it is a very small volume, it should be mixed immediately
upon addition. High local salt
concentrations will cause some nuclei to lyse.
10.
Allow the
nuclei to extract for 30 minutes with continuous gentle mixing. I use a tube rotator in the cold room.
11.
Pellet the
extracted nuclei by centrifuging 30 minutes at 25000 X g. Draw off the resulting
supernatant. This is the
nuclear extract.
12. Bring the extract up to 100 microliters with a solution that
consists of equal parts high and low salt buffer, if the volume is less than
100 microliters. Dialyze for 45
minutes. Centrifuge the dialysate
20 minutes at 14,500 rpm in microfuge.
Discard the pellet. Remove
5 microliters from the supernatant to due a protein concentration assay on, and
freeze the rest at –80.
Low
salt=20mM KCl
High
salt=1.2M KCl