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Fluorescent Protein Expression in Yeast

Kirk Leifso leifsok at uvic.ca
Fri Jul 26 17:10:54 EST 2002


Hello,

My name is Kirk Leifso and I am a Master's student at the University of
Victoria.  I am working with Kluyveromyces lactis and am studying at carbon
catabolite regulation.  I am using my promoter to drive expression of
fluorescent proteins (ECFP and EYFP from Clontech) that have been tagged
with a secretion signal in the hopes that I can sample the culture media and
detect levels of fluorescent protein.  I have already confirmed that the
fluorescent proteins are indeed being secreted into the culture media by
SDS-PAGE and subsequent western blot using Anti-GFP antibodies (also from
Clontech).  I am trying to detect fluorescence using a PTI QM-2
spectrofluorometer.

As of yet I have not been able to detect fluorescence, and am wondering if
anyone knows (or has a guess) at the lower limit of detection for these
fluorescent proteins (in terms of ng/ml or some other concentration).  As
well are there any conditions that I should be concerned about while growing
up my cells (I am just growing my yeast in non-baffled Erlynmeyer flasks)
and should I expect a loss in fluorescence with storage of protein samples
over time?

Any thoughts or suggestions would be helpful.

Thank you,
Kirk Leifso
Department of Biology
University of Victoria
Victoria, British Columbia
Canada
leifsok at uvic.ca


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