GFP in yeast (fwd)/re: fixing cells for FACS

'Mike' Michael J. Moser moser at U.WASHINGTON.EDU
Tue Nov 11 19:16:41 EST 1997


Dear GFPers,

I received this message a few days after a posting I made about fixing
cells for FACS.  I wanted to post Arle Kruckeberg's message and my reply
for general interest, comments and etc. 

Mike Moser                                            Tel: 206-543-6585
UW Department of Pathology                            FAX: 206-543-3967
Box 357705                                       moser at u.washington.edu
Seattle, WA  98195                 http://weber.u.washington.edu/~moser

---------- Forwarded message ----------
Date: Mon, 10 Nov 1997 18:26:18 +0100
From: Arle Kruckeberg <arle at chem.uva.nl>
To: moser at u.washington.edu
Subject: GFP in yeast

Hi Mike,

I've seen a few of your posts about yeast expression of GFP, and thought
I'd put a technical problem to you.

We are expressing fusions of GFP with yeast plasma membrane
proteins...works great, when we view the living cells, but when we fix
GFP-expressing cells and process them for immunofluorescence microscopy the
GFP signal is significantly attenuated. We use more or less the conditions
described in John Pringle's article in MetEnz vol 194, and have varied the
time and temp of fixation; we even tried a new fixative from Amresco,
HistoChoice (no good). The only difference between what we do and what you
describe in your post is that in your protocol the formalin exposure is so
short, and that you incubate in PBS without formaldehyde before further
processing (and you still presumably get good cross-linking). Are prolonged
exposures to formaldehyde deleterious? Also, GFP is said by Clontech to be
sensitive to strong reducing agents. The Pringle mounting medium contains
p-phenylenediamine to minimize photobleaching. This is, I think, a reducing
agent. Could it be the culprit of our reduced EGFP signal? Or do you use it
too?

Thanks,
Arle
ps Hows the weather in my old home town?
------------------------------------------
Dr. Arthur L. Kruckeberg
E.C. Slater Institute/BioCentrum Amsterdam
The University of Amsterdam
Plantage Muidergracht 12
1018 TV Amsterdam
The Netherlands
+31 +20 525 5069 (office)
+31 +20 525 5127 (lab)
FAX +31 +20 525 5124
e-mail arle at chem.uva.nl





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