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.



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Department of Molecular Genetics and Microbiology
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-----Original Message-----
From: Kendra Hyland [mailto:kendrah@discoverygenomics.net]
Sent: Fri 12/7/2007 9:53 AM
To: cyto-inbox
Subject: GFP loss after fixation and permeabilization
 
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
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Kendra Hyland, Ph.D.
Discovery Genomics, Inc.
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