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[Fluorescent-proteins] Re: Quenching Fluorescent Proteins

Dr Mephesto via fluorpro%40net.bio.net (by dnhkng from gmail.com)
Tue Jun 17 08:07:00 EST 2014


Can you just bleach out the RFP with strong monochromatic light? According to Tsien's paper (http://www.tsienlab.ucsd.edu/Publications/Shaner%202005%20Nature%20Methods%20-%20Choosing%20fluorescent%20proteins.pdf), dsRED have very low photostability.
-Dave


On Friday, November 21, 2008 11:55:09 AM UTC+1, Confused Dave wrote:
> Hello,
> 
> I'm looking for a way to reliably inactivate fluorescent proteins
> (EGFP and dsRed) without harming epitopes for immuno.
> 
> A little background:  I'm electroporating inducible constructs into
> the chick embryo.  We use dsRed as the electroporation control, and
> EGFP as a marker for activation of our doxicyclin-inducible construct.
>  We want to use immunofluorescence to on these samples.  We currently
> use 4% formalin to fix, washes with PBS+tween, usually ethanol
> dehydration for storage, and sucrose-agar embedding for sectioning,
> and the fluorescence still comes up strong.  Obviously, imaging with
> epifluorescence with a green and red channel, we can't add another
> fluorophore.
> 
> Best case would be a way to specifically kill the RFP leaving the
> green intact, although as long as we can recover the GFP with an
> antibody, this isn't a problem.  Also looking for something that won't
> damage our epitopes to badly!
> 
> Thanks,
> Dave


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