GFP

Dan Zabetakis dan at cubsps.bio.columbia.edu
Mon Jun 12 23:17:16 EST 1995


In article <3rhljk$g4u at mercury.hgmp.mrc.ac.uk>,
**** <c-talbot at nimr.mrc.ac.uk> wrote:
>Does anyone have any info about expression of the GFP protein in the Yeast S. cer.
>
>I have a functional construct but no fluorescence???

  My information is incomplete, but I have had some experience with GFP.
My conclusions are these:

  1) Your scope matters. GFP signal can vary a lot depending on your
scope and filter setup, even if it theoretically is the same as published
procedures. If you know someone who is using GFP well, try thier scope.

  2) Yeast are green. It seems that the GFP has overlapping excitation and
emmission spectra. There is often a high background in yeast generally.
If you protein is localized to a discrete compartment, you might see it
while if your protein is cytoplasmic, you may be out of luck. Reducing
the background may reduce your signal too low.

  3) Fixation and other techniques are not necessary. In fact, I sometimes
had better result looking at cells in media than after washing with water.
I think maybe the media quenched some of the background, but that's 
probably bull.

  4) Seeing GFP in cell extracts is hard. Not your question, but I'll
say it anyway. The flourescence of a yeast extract is so high that there
was little hope of detecting the GFP specific signal.

DanZ

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