Subject: Do I have cell wall fraction of yeast

From: [email-masked] (Dr. C. Mody)
Date: Mon, 13 Feb 1995 18:13:28 -0700

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
[email-masked]

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From: [email-masked] (Jessie Micales)
Date: Thu, 16 Feb 1995 16:24:32

>[email-masked]

You may want to redisperse and wash the pellet in buffer several times to 
remove any contaminating cytosolic material.  As for assays, good 
question!!!  There are lots of commercial protein assays available (check 
BioRad, for example), but I believe that cell walls can also contain some 
protein on their own (the concentration would probably be fairly low, 
however).  Many fungi also produce an extracellular mucilage (usually 
glucan) that can contain many extracellular proteins.  This glucan is 
actually an extension of the cell wall.  It can be removed from the cells by 
washing them in buffer before you break open the cells.  A little detergent 
in the buffer (such as Triton X-100) can also help remove the mucilage.

Good luck.  Please let me know if anyone e-mails you any better 
protocols!

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From: [email-masked] (SMazzer)
Date: 27 Feb 1995 19:59:28 -0500

>It can be removed from the cells by 

Be careful using this method.  Triton X-100 (as well as other detergents)
tend to give false-positive results in many commonly-available protein
assays.  

Paula Mazzer ("[email-masked]")