I had the same problem with GFP expression, and it turned out to be the pH
of the formaldehyde. At that time, I had started using a sub-standard grade
of formaldehyde, and when I checked the pH of the solution (in PBS) it was
nearly ph 3!
I adjusted the pH, then repeated the experiment, and suddenly my
fluorescence reappeared. After that, I purchased EM-grade formaldehyde and
have never since had the problem.
Good luck.
JDT
+++ +++ +++ +++ +++ +++ +++ +++ +++
Julie Davis Turner, Ph.D.
Postdoctoral Fellow
Centers for Disease Control and Prevention
NCID, DASTLR, Tuberculosis Branch
Pathogenesis Laboratory
Atlanta, GA 30333 U.S.A.
-----Original Message-----
From: Elena Soriano [mailto:E.Soriano@iam.boku.ac.at]
Sent: Thursday, March 29, 2001 7:59 AM
To: cyto-inbox
Subject: GFP in bacteria
I´m trying to measure the GFP expression in bacteria, and I´ve got a nice
signal, but not for all samples, though they have all been treated the
same. We fixed them with 4% formaldehid in PBS at 4°C.
I´ve been told that the GFP is quite small, could it be that it gets
through the fixed cell wall?
Could somebody give me an advice? Does it exist a better kind for fixing
the bacteria for GFP analysis?
Thanks in advance!!
-----------------------------------------
Elena Soriano
c/o Institut für Angewandte Mikrobiologie
Univ.f.Bodenkultur Wien
Tel: +43 1 36006 6241
Fax: +43 1 36 97 615
http://www.boku.ac.at/iam
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