Preparation of nuclear extract: How to lyse the cells?

Martin Latterich mdhouse at MENDEL.BERKELEY.EDU
Wed Mar 22 22:01:05 EST 1995


>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?

Julie,

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.

Martin



Dr. Martin Latterich
Howard Hughes Medical Institute and
Dptm. of Molecular and Cell Biology
University of California,
Berkeley, CA 94706
USA
Tel.:  (510) 624-6171 (W)
FAX:   (510) 642-7846
e-mail: micro at mendel.berkeley.edu
        latteric at mendel.berkeley.edu





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