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Sinbce you are (as I read you) expecting co-localization it may
be that you are getting FRET is your co-transfected cells.  If this
is the case you will get excitation of the DS-red by blue in the
co-transfected cells but mainly by green in cells that lack GFP.

                                                        Guy


>  hi,
>    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.
>
>    please kindly send in your suggestions.
>Thaking you,
>Shagufta Rehman
>Ph.D. student
>Dept of Pathology
>AIIMS, New Delhi
>India.
>
>
>
>
>
>
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