Preparation of nuclear extract: How to lyse the cells?
rout at rockvax.rockefeller.edu
Fri Mar 24 23:11:12 EST 1995
In article <9503240054.AA07334 at mendel.Berkeley.EDU>,
mdhouse at MENDEL.BERKELEY.EDU (Martin Latterich) wrote:
> Hi Mike,
> thanks for the info - it's been some time since I last attempted the prep.
> I think the nuclear prep method using the modified Kilmartin method is
> definitely the method of choice when making yeast nuclei because it's yield
> is superior to other protocols. However, when omitting Tx-100 from the prep
> some strains will produce nuclei with sheets of plasma membrane attached -
> when I used Tx-100 at a dilute concentration, I gotten perfect nuclei.
> However, the properties of the nuclear envelope, though morphologically
> intact, suggested that the detergent had permealized the nuclear ER
> membrane (sorry ;-)). Based on personal experience, it depends very much on
> the strain one is using and what one needs the nuclei for.
> Hope this helps,
Thanks for the reply info - I agree, we've also certainly noticed strain
dependent results. Re NE properties - we've been working with highly
enriched preps of "functional" NE (i.e., ER post-translational
translocation still works) but we've only tried so far -
i) no detergent during sphero lysis
ii) mainly the (very amenable) Saccharomyces uvarum strain.
Problems with other strains (limited experience) seem to manifest as
reduced NE yield. The nuclei prep seems to work best if you can use a
diploid strain of whatever you want to use, and I would strongly recommend
the high pH / DTT pretreatment, instead of the detergent, with other
strains. (It's not part of the published protocol). It really seemed to
help in the few times we did this prep with other strains. I intend to
play around a bit with the conditions for other strains in the near(ish)
future. I'll keep you (and this noticeboard) posted if I turn up anything
new. Do you know if the method of lysis (Polytron vs. Potter / Dounce
etc.) can produce different results with different strains? I wonder if
using digitonin, or something else, instead of TX100 would be
better......? Anyone out there tried?
Hope this helps (and makes sense!!)
All the best
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
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