In article <4a6k2o$g4o_001 at uovs.ac.za>, gnhmghjp at med.uovs.ac.za (G H J
Pretorius) wrote:
> Dear Netters,
>> We are trying to isolate yeast nuclei for footprinting purposes, since
on bandshifts we get a fairly weak complex using crude extracts.
> We have encountered a number of problems:
> 1. The spheroplasts wouldn't lyse in either 18% Ficoll 400 or 8% PVP
40, even after extensive homogenisation with a Polytron. They are real
spheros, since they lyse in water
> and have no cell wall under the microscope.
> 2. We use the Percoll gradient method, but nearly everything bands in
the middle where one would expect the nuclei, but under the microscope
> they look like spheroplasts. DAPI staining shows a strong signal
inside, but there are still lots of "cytoplasm" around. They are also the
same size
> as the sheroplasts.
> 3. We have followed cytoplasmic lysis with ADH activity, but the
majority is still associated with our "nuclei".
> 4. Are they really so hard to lyse?
>> Cheers,
> Oubaas Pretorius.
How are you digesting the cells? This can often be a problem. Spheros
can still have a residue of cell wall, that doesn't really show up in the
light microscope, and doesn't stop them from popping in dH2O, but still
interferes with polytron lysis. Try -
- pre-treating the cells in 100mM Tris-Cl pH 9.4, 10mM DTT, 10 min, room
temp; then wash 1x dH2O, 1x 1.1M sorbitol prior to digest.
- digest in 1.1M sorbtiol, 1:10 glusulase, 1:100 1% zymolyase 20T, 1:100
mutanase (SP299, Novo), ~3 hr, 30C.
- If your protocol can stand it, 0.025% Triton X-100 in the 8% PVP lysis
solution (~30ml lysis solution per 6g wet wt spheros). We've found no
apppreciable loss of nuclear markers (and you usually see lots of intact
vacuoles floating around in the lysate, which are more fragile than
nuclei) - I think basically nearly all the Triton gets mopped up by the
plasma membranes (...?)
- the sucrose-PVP nuclear enrichment gradient may give you better removal
of cytoplasmic contaminants (aggregates of cyt rubbish tend to shear off
the nuclei in the high viscosity gradient during the centrifuging).
Good luck! Hope this helps,
Mike.
--
Mike Rout 212 327-8098 (lab)
Laboratory of Cell Biology 212 327-8660 (fax)
The Rockefeller University/HHMI
1230 York Ave.
New York, NY 10021
