From what I understand: for energy transfer to occur the emission spectra of the GFP would have to overlap the excitation spectra of the DS RED. There's a certain formula that's applied but I don't have it right now but I'm sure someone will. I However the DS red is also excited to some degree at the same wavelength as the GFP so I don't believe you could actually measure FRET with these two. The better option is CFP and YFP but you need a 410nm or 442nm laser for this. This is what you would see if using the 410nm laser to excite CFP : in the 410nm pathway: emission filter 488nm- you see all CFP positives, emission filter 547nm -all FRET positives i.e if energy transfer occurred. Otherwise no signal as the YFP is not excited at this wavelength. To see double positives you need 2 lasers -410nm for CFP and 530nm for YFP, and plot CFP from laser one against the YFP signal coming of laser two. Could you please post responses as I am interested in this too. regards Ann At 14:19 12.08.02 -0500, you wrote: >Howdy, > >I am having a little problem understanding this whole energy transfer >issue. Say you had a GFP protein and another protein fused with DS >Red. Also say that these two proteins are supposed to interact with each >other. The energy should be transferred from high energy to low >energy. Or GFP to DS Red. >So her comes the questions. Does this strictly result in a loss of GFP >fluorescence and a gain in DS Red fluorescence? One of our users went to a >lecture that stated that you actually can get a change in emitted >wavelength(Unpublished data). In the above example the DS Red would now be >Yellow in emission. Is this True? Could this be true for confocal but not >for Flow? Do we now need to use a different dichroic to find the double >positive cells? Am I totally off on this whole issue? Be nice!!! > > > Thanks in advance for the help, marv >
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