Does anyone have experience doing BFP/GFP or CFP/GFP FRET in transfected cells using a confocal microscope with LEICA software? We'd like to get that geared up, but in our naivity have a basic conceptual problem. If we excite the BFP with its UV excitation, then we imagine its emission ought excite GFP that is close enough by, and then we can monitor emission of the GFP as the indicator of energy transfer. (Why not alternatively monitor quenching of the BFP emission by GFP as the indicator of FRET?). However, the BFP emission itself does have an overlap into the GFP emission, so that has to be subtracted out of the 'GFP emission.' The problem is how to estimate the amount of BFP emission that overlaps into the GFP emission range. We can just use some BFP-only transfected cells, since there is great variation from one cell to the next on the intensity of the BFP emission in BFP-only transfected cells. Can we do something alternative, such as scan the shape of the BFP emissionin BFP-only transfected cells, establish an empirical distribution of BFP emission vs. wavelength, and then derive a statistic on, given a certain peak value of BFP emission in BFP/GFP cotransfected cells, we know that x-amount of BFP emission value must be happening in the region of emission overlap between BFP and GFP? That calculated x-amount can then be substrated from the observed 'GFP (+BFP tail) emission'? Help! Is it feasible to use a CFP/GFP set? Grace Jones
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