q

David R. Nelson dnelson at utmem1.utmem.edu
Thu Dec 22 16:59:58 EST 1994


In article <1994Dec21.194324 at crick.rz-berlin.mpg.de>,
andre at mpimg-berlin-dahlem.mpg.de (CHAZ) wrote:

> Does anybody have suggestions for preparing mitochondria
> from S. cereviseae?  I have been making spheroplasts with
> zymolase, but I have trouble breaking the cells open-
> I tried a tissue homogenizer, but even after 30 strokes
> the yeast seem to be quite happy and intact.  Any other
> ideas?   Thanks in advance for suggestions.
> 
> C. Andre

We use 0.5mm glass beads (acid washed) and mix the spheroplasted cells with
the beads to make a paste about the consistency of peanut butter (rather
thick). Then we grind these with a small glass rod in the bottom of a
plastic centrifuge tube (SS34 rotor tube, or JA20) A couple minutes of
grinding usually releases the mitochondria.  We also used glusulase in the
spheroplast step.
Keep it all cold.  See J. Mol. Biol. 230 1159 (1993) methods section d
-- 
David R. Nelson
Assistant Professor
Dept. of Biochemistry
University of Tennessee, Memphis



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