Re: GFP/RFP quenching

Posted by Phillips, Thomas E. on Mar 04, 2009; 4:27pm
URL: http://confocal-microscopy-list.275.s1.nabble.com/GFP-RFP-quenching-tp2422621p2423882.html

Paraformaldehyde has no methanol. Formalin does and therefore it is
incorrect to use the terms interchangeably.  

Thomas E. Phillips, Ph.D
Professor of Biological Sciences
Chair, MU Faculty Council
Director, Molecular Cytology Core
2 Tucker Hall
University of Missouri
Columbia, MO 65211-7400
573-882-4712 (office)
573-882-0123 (fax)
[hidden email]

http://www.biology.missouri.edu/faculty/phillips.html
http://www.biotech.missouri.edu/mcc/


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of [hidden email]
Sent: Wednesday, March 04, 2009 9:43 AM
To: [hidden email]
Subject: Re: GFP/RFP quenching

Scott,

Regular paraformaldehyde will quench the GFP signal because of the
methanol that forms in it. We use methanol free formaldehyde, 10%,
ultrapure from Polysciences.

Any alcohol will quench the GFP signal.

Since this formaldehyde comes already in solution, it also saves you
the time and trouble of preparing the paraformaldehyde yourself. We
just dilute it to 4% in PBS pH 7.4 with no calcium or magnesium, and
use it immediately. I always make up only what I need, and don't try to
store the diluted formaldehyde.

Deb Martinson
Research Specialist
Internal Medicine Imaging Core
Division of Cardiology
Department of Medicine
Emory University School of Medicine
101 Woodruff Circle WMB 303
Atlanta, GA 30322

404-727-3712


Quoting Scott Howell <[hidden email]>:

> List,
>
> We have had some issues here with a lab where the GFP/RFP brightness
in
> fixed cells has become extremely variable. Like night and day.
Wondering

> if it may be related to the pH of their paraformaldehyde fix? Does one
> particular pH seem to work best?? Any other ideas? Thanks.
>
> Scott J. Howell, Ph.D.
> Manager, Imaging Module
> Visual Sciences Research Center
> Case Western Reserve University
> 2085 Adelbert Rd.
> Institute of Pathology Room 106
> Cleveland, Ohio 44106
> 216-368-2300
> http://www.case.edu/med/vsrc/
>
>
>