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shagufta rehman wrote:

>     I am using ERDsRED (from clontech)with EGFP tagged
> protein in a co-transfection for a co-localization
> study. The expression of my EGFP tagged protein is
> good enough to be easily captured by confocal
> microscope (as observed in separate experiments as
> well as a control to monitor the expression of EGFP
> tagged protein while doing co-transfection). However,
> after the co-transfection it is difficult for me to
> identify cells with both green and red fluorescence.
> In an epifluorescence microscope when i look for the
> co-transfected cells, under the blue light, they
> appear orange, which could be because of only the red
> fluorescence as similar orange fluorescence I observe
> with the control i.e. only ERDsRed transfected cells.
> In the co-transfected cells I do not see any green
> fluorescence. Is it that the fluorescence from ERDsRed
> is masking the expression of EGFP tagged protein? also
> why a red protein (ERDsRED)is getting excited by a
> blue light.
>

In your epifluorescence microscope, you probably have
a broad low pass emission filter with your blue excitation
which will pass both DsRed and GFP emission. DsRed has a
very broad excitation spectrum and can certainly be excited
with blue light (take a look at the spectra on
http://home.ncifcrf.gov/ccr/flowcore/DsRed.htm)

If you want to isolate GFP emission, you must use a narrow band emission
filter that cuts off all emission of wavelengths over 540nm.
Even if there is FRET (as suggested in one response) you should still see
plenty of GFP emission. After all, a FRET efficiency of 50% would be huge
(CFP/YFP has been measured around 15%-20%), and that still leaves half
of your GFP emission.

Also, you must be sure your DsRed (or some of it) has not turned green
again (it matures from green to red). See
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=17282
for a discusison about what makes it red and how it can revert.
I also saw something on the "greening" of DsRed using 2-P excitation.
If a small precentage of DsRed is not "matured". then you will get
GFP like emission from it, which you may mistake for GFP. This can happen
even though it appears orange when you look at it. You can check these
things with narrow band filters.

--aryeh
--
Aryeh Weiss
School of Engineering
Bar Ilan University
Ramat Gan 52900 Israel

Ph:  972-3-5317639
FAX: 972-3-5340697