Katryn, could this be due to resonance energy transfer?
GFP converts blue light to green and excitates TRITC and alike dyes.
This cause the GFP signal to become dim at colocalization spots.
Cheers, Wo
> Katryn Sunn wrote:
> To: fluorpro at net.bio.net> From: k.sunn at garvan.unsw.edu.au (Kathryn Sunn)
> Subject: COLOCALISATION WITH GFP
> Date: 10 Oct 1999 22:52:34 -0700
> Dear All
>>> Has anyone done a lot of colocalisation studies using GFP tagged
> transfected constructs and a red-tagged antibody? If so has anyone
> ever had the problem of the GFP green signal losing its fluorescence
> after permeabilisation? Does anyone know any handy hints to get
> around this at all?
>>> Thanks for your help
>>> Kate
>>> ___________________________________________________________________
> Kathryn Sunn BSc (Hons) Postgraduate Student Bone and Mineral
> Research Program Garvan Institute of Medical Research 384 Victoria
> St, Darlinghurst St Vincent's Hospital Tel: (02) 9295 8260 Sydney
> NSW 2010 Fax: (02) 9295 8241 Australia email:
>k.sunn at garvan.unsw.edu.au> ___________________________________________________________________
>>>> --------------------------------------------------------------------
> -------- ----
>> Hi Katryn
> It proabely depends on your procedure. Whitch procedure are you
> using? I have immunostained the transferrin receptor with texas-red
> in GFP-fusion expressing primary muscle cells, with out any problems
> with the EGFP fluorescence. I use fist 2% paraformaldehyde to fix
> followed with saponin for permeabilisation and finally the
> antibodies with saponin. I mount in vectashield. I have not seen any
> decrease in EGFP fluorescence on specimen even after 1¢ year in the
> fridge.
> Med venlig hilsen Hans
>>> Hans P.M.M.Lauritzen, B.Sc., M.Sc., Phd-student.
> Copenhagen Muscle Research Center, The Panum Institute 12.4
> Copenhagen, Denmark
> E-mail: H.P.Lauritz at mfi.ku.dk> Phone: +45 35327375 or +45 35327436 or +45 35327430
> Fax: +45 35327420
-----
usual disclaimers apply
-----
Dr. Wolfgang Schechinger
Pathobiochemistry Dept.
University of Tuebingen, Germany
email: wgschech at med.uni-tuebingen.de
wwWait: http://www.medizin.uni-tuebingen.de/~wgschech/start.htm
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*unsolicited mail is *NOT* appreciated
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