GFP and FITC

Bernard Murray bernard at elsie.nci.nih.gov
Thu Feb 6 15:16:42 EST 1997


In article <32F97341.6EB5 at probes.com>, dick at probes.com says...
>
>One can distinguish GFP and fluorescein in a fluorometer by differences
>in their their relative excitation spectra but not by their emission
>spectrum  The GFP excitation spectrum is similar to its shorter
>wavelength absorption spectrum.  If you are talking about microscopy it
>is not possible to resolve these by ordinary means.  If you have both
>present it MAY be possible to measure the emission of the sample in a
>digital imaging instrument at a neutral pH then the acidify the medium
>to below pH 6.  Because the fluorescence of fluorescein is partially
>quenched in acid, its intensity will decrease, whereas I would not
>expect that of GFP to be affected by pH.  This could be done on a
>pixel-by-pixel basis and then to results color colded in contrasting
>pseudocolors.
>
>Dick Haugland, President
>Molecular Probes
>dick at probes.com

Nice ideas.  It may also be possible to do it the other way around and
quench the GFP fluorescence.  According to the Clontech blurb that
accompanies their vectors you can accomplish this with phenoxypropanol,
or reducing agents (Na2S2O4 or FeSO4), or hydrogen peroxide, or thiol
active agents such as DTNB.  If fluorescein can survive any of these
then this may be another way to go.
	The pamphlet also says that GFP fluorescence *is* decreased at
acid pH so this may not give the selectivity you are looking for.
Clontech's document (PT2040-1) is available on their web site.
		Bernard

PS: You may also want to cross-post to bionet.molbio.proteins.fluorescent

[I have no affiliation with Clontech]

Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)




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