FW: GFP loss after fixation and permeabilization

------ Forwarded Message
From: "Gerstein, Rachel" <Rachel.Gerstein@umassmed.edu>
Date: Fri, 7 Dec 2007 15:54:40 -0500
To: cyto-inbox
Conversation: GFP loss after fixation and permeabilization
Subject: RE: GFP loss after fixation and permeabilization

hi 

"Perhaps the GFP fluorescence is quenched after treatment with the
detergent?"
yup. it probably changes the structure enough to change fluorescence.

I would sort the (live) cells into GFP+ and GFP- and then do your
intracellular stain.



=======================================================
Rachel M. Gerstein, Ph.D.
Associate Professor
Department of Molecular Genetics and Microbiology
Graduate Program in Immunology/Virology
University of Massachusetts Medical School
55 Lake Avenue North
Worcester, MA 01655-0002
(508) 856-1044
(508) 856-5920 (FAX)



-----Original Message-----
From: Kendra Hyland [mailto:kendrah@discoverygenomics.net]
Sent: Fri 12/7/2007 9:53 AM
To: cyto-inbox

Hello,

I am new to the list and I was looking at past postings regarding my
problem.  I want to stain for an intracellular protein and detect GFP+
cells at the same time.  I fixed the cells with BD Cytofix (4%
paraformaldehyde) and there was no difference in % GFP+ cells in fixed
and fresh cells.  But if I fix followed by permeabilization with 0.4%
saponin/ 35% FBS/ PBS buffer, then the %GFP+ cells is reduced by about
half.

I find it hard to believe that the GFP is leaking out of the cells, as
it is larger than some cytokines which can be detected intracellularly
(GFP is 27 kDa, mouse IFNgamma is 18 kDa and mouse IL-2 is 19 kDa).
Perhaps the GFP fluorescence is quenched after treatment with the
detergent?

 From past postings, it sounds like I could alter my transfected
construct so the GFP is membrane bound or possibly use an anti-GFP
antibody.  Does anyone have another solution?  Would other detergents,
such as Triton X-100 or Tween20, not impact the GFP fluorescence as
much as saponin?

Thank you,
Kendra
_________________
Kendra Hyland, Ph.D.
Discovery Genomics, Inc.
614 McKinley Place N.E.
Minneapolis, MN 55413

ph/ 612-656-4485 (office)
fax/612-379-6580


http://www.discoverygenomics.net/





------ End of Forwarded Message