We (and other companies) do have both polyclonal and monoclonal antibodies to GFP and also sell an Alexa Fluor 594 direct conjugate of rabbit anti-GFP http://www.probes.com/servlets/product?region=Select&item=21312 as well as the green-fluorescent Alexa Fluor 488 direct conjugate http://www.probes.com/servlets/product?region=Select&item=21311 However, I would expect that any polyclonal anti-GFP antibodies would likely recognize both folded, and thus fluorescent, forms of GFP and incompletely folded, and thus nonfluorescent forms of GFP. We have shown in one experiment that a red fluorescent direct conjugate of anti-GFP (directed toward mitochondrial expression) yields red-fluorescent unfolded GFP. If the folded and unfolded GFP are colocalized, one might observe yellow fluorescence or, if they are spatially resolved (as in our single experiment) a mixture of green and red fluorescence. In any case this could be a complicating factor in tissue expression studies too. It may be possible to do the same experiment with an R-PE anti-goat (or rabbit) secondary antibody but would increase the likelihood of FRET in the case of colocalization (due to the higher spectral overlap and extinction coefficient of the R-PE acceptor), resulting in only red fluorescence of the colocalized folded GFP. I am not certain whether the selectivity for folded versus unfolded GFP also applies to our monoclonal anti-GFP antibodies. "Richard K. Meister" wrote: > Hello, everyone: > > I have a client who needs to detect GFP in fixed tissue sections (various > animal tissues) and he is concerned that the fixation process might effect > the fluorescence of the GFP. I haven't done GFP in tissue, but I have > routinely with fixed flow specimens - and they work just fine. > Nevertheless, he is concerned that he might miss some GFP-labelled (but > non-fluorescent) sites. So, he is thinking of coming in with a goat > anti-GFP antiserum, followed by an anti-goat 2nd antibody:PE. > > So, he has a few questions. First, is there a particular fixation protocol > that he should followfor GFP-labelled tissue? > > Second, what anibodies do you recommend? And what is your staining > protocol? Any other tips? > > Thanks in advance. > > Rick Meister > * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * > * Richard K. Meister Email: meister.1@osu.edu * > * The Ohio State University Voice: (614) 292-9716 * > * Dept. of Veterinary Biosciences FAX: (614) 292-6473 * > * Cytometry Instrumentation Lab * > * 1925 Coffey Road * > * Columbus, OH 43210 U.S.A. * > * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *
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