Do I have cell wall fraction of yeast?

Dr. C. Mody cmody at acs.ucalgary.ca
Mon Feb 13 20:12:52 EST 1995


Can anybody tell me how to be resonably sure that I have separated the
cell wall and cytosolic fractions of Cryptococcus neoformans. C.
neoformans was grown in Sabs broth, killed with heat (autoclaved), washed
and about 10^9 organisms placed in a glass bead mill [Bead Beater®]
(volume of organisms 75 ml, beads 100 ml, buffer to fill chamber). Rotated
one minute on, one minute off for 60 minutes on ice.  Disruption was
complete by microscopic exam. A slow spin was done to remove debris.  The
preparation was spum at 13,000 xg for 30 minutes, and the pellet and
supernatant was collected. My reading would indicate that the pellet
should contain the cell wall, the supernatant the cytosol.  How can I be
more sure? Is there a simple assay that can be done to tell that my cell
wall preparation has minimal contamination with cytosolic proteins and
vice versa?

Thanks
Chris Mody
cmody at acs.ucalgary.ca



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