[Yeast] caspase assay protocol

Amy Guan xguane at gmail.com
Tue Mar 7 16:43:15 EST 2006


Dear List,

Hi, I'm a high school student conducting a science fair project on the
presence of the YCA1 caspase in P. pastoris (has been discovered in
Saccharomyces).  I have asked around but nobody seems to know how to measure
the activity/presence of this caspase in yeast and upon searching for
methods, I have come across flow cytometry, spectrophotometry, and
fluorimetry techniques however, all seem to be suited for mammalian cells
and there aren't any step by step procedures.  Does anyone know of a good
caspase assay protocol I can use to detect YCA1 activity in P. pastoris?

Additionally, I'm also trying to induce nitric oxide production by inducing
the nitric oxide synthase enzyme in this yeast however, this again is
usually done in mammalian cells and NO induction is achieved by addition of
a wide variety of cytokines and lipopolysaccharide.  Upon searching for
literature, no results have come up regarding yeast sensitivity towards LPS
addition and it is unlikely for LPS to cross the yeast cell wall.  Do you
know of a NO promoting drug that I could use in yeast or possibly if yeast
could be sensitive towards LPS induction?

Thanks.

Amy.
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