Preparation of nuclear extract: How to lyse the cells?
rout at rockvax.rockefeller.edu
Thu Mar 23 19:11:23 EST 1995
In article <9503230300.AA29361 at mendel.Berkeley.EDU>,
mdhouse at MENDEL.BERKELEY.EDU (Martin Latterich) wrote:
> >Hi everybody!
> > I'm having a hard time trying to reproduce the protocole of Wang et al (in
> >Biochemistry 1992- vol.31 p.3694-3702). My cells are just not breaking open!
> >In this protocole, they say that, after a digestion with zymolase (1mg/g of
> >cells), the spheroplast preparation "was lysed in a motor-driven homogenizer
> >with 10 strokes "(!!!). They do not talk about the device itself and, so, I
> >tried to break them using a polytron (maximum power, more than 5min...) from
> >Brinkmann cie ( model Kinematica): it just do not work!
> >Do somebody know what type of apparatus they used? May I ask for tricks and
> >hints from those how have successfully overcome the troubles of making yeast
> >nuclear extracts?
> in my experience, the best (and unfortunately lowest yield method) of
> isolating morphological nuclei and subsequent nuclear extracts is that of
> Aris and Blobel (1991) Meth. Enzymology 194, 735-749. The protocol to use
> somewhat depends on what you are interested in, and there are many other
> excellent protocols around that are very well suited for making nuclear
> pore preps - however, they rely on the addition of non-ionic detergents
> which can disrupt nuclear integrity. The best structural integrity of the
> nucleus is maintained following the Aris and Blobel protocol, making this
> the protocol of choice for preparing nuclear extracts. Drawbacks are that
> you need large volumes of yeast culture, and it is low yield (1 %). Also,
> you need to have a Wheaton Dounce Homogenizer as homogenization is crucial
> to yield and integrity.
> Dr. Martin Latterich
> Howard Hughes Medical Institute and
> Dptm. of Molecular and Cell Biology
> University of California,
> Berkeley, CA 94706
> Tel.: (510) 624-6171 (W)
> FAX: (510) 642-7846
> e-mail: micro at mendel.berkeley.edu
> latteric at mendel.berkeley.edu
Just a quick note - It's a little confusing, because this prep has gone
through a number of minor changes, but the addition of Triton X-100 to the
spheroplast lysis step of the Kilmartin nuclear isolation prep mentioned
above is not absolutely necessary and was never done in the isolation of
yeast nuclear pore complexes (as it sez in the Rout and Blobel paper "fine
print"! ;-) ) - therefore it should be fine to use this prep for the
preparation of your extracts. Nuclear yields are usually 60 - 80%, and it
can be scaled down. The structural integrity of these nuclei seems fine
when examined by EM, and numerous nuclear/ nucleolar markers coenrich
absolutely by quantitative immunoblotting (we're publishing this soon,
hopefully! Some of these markers can be seen in the R and B paper).
Actually, careful use of Triton in the prep (as described originally)
probably won't affect nuclear integrity either - the idea is, the very
small amount of detergent gets "mopped up" by the plasma membrane,
weakening it so the spheros lyse more easily. (Honest). Avoiding the
detergent altogether, you might try the 100mM Tris-Cl pH 9.4, 10mM DTT, 10
min, 30 C treatment of cells prior to spheroplasting, if your cell strain
is being stubborn. A very detailed description of this method (large
scale) can be found in "Cell Biology: A Biology Handbook, Vol. 1"
Academic Press, 1994, Ed. Julio E. Celis pp 605-612 (except, don't add the
Hope this is of some help,
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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