In article <favre-1610961448420001 at 132.206.101.173>
favre at medcor.mcgill.ca (Daniel Favre) writes:
> I have a GFP-fusion protein that can be induced with galactose. My aim is
> to generate a pool of this protein, inhibit it's production by growing the
> cells in glucose-containing medium, and then analyze the localization of
> the protein by fluorescence.
> Questions: is a 30 minutes galactose induction sufficient? How long does
> it take to repress the expression after the cells have been shifted to
> glucose-containing medium? In this respect, what are indeed the optimal
> timings?
>> Thank you very much for your attention and comments
> Daniel
Hi Daniel,
I doubt that you will get very much of your protein expressed within 30
minutes, but you could do a time-course to see how long it takes to get
enough to see (analogous to the "pulse" phase?). The glucose shift
should turn off expression quite rapidly; for example GAL2 mRNA is
undetectable within 3 minutes of a glucose pulse (Sierkstra etal Yeast
8:1077).
Have fun!
Arle
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a.k.a. Dr. Arthur L. Kruckeberg
Department of Biological Chemistry
UCLA School of Medicine
Los Angeles CA 90024
(310) 825-8363 FAX (310) 206-5272
e-mail kruckeba at biovx1.biology.ucla.edu
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