FRET Introduction (Page 6)
Let's return to the FRET experiment:
We have two proteins tagged with YFP and CFP and co-expressed in the cell. We acquire images from the 3 channels in the order shown. In practice we have found that the order of acquisition is important because YFP rapidly bleaches when exposed to the CFP excitation light. From the CFP and YFP channel data we calculate the total fluorescence expected in the FRET channel from spillover:
FRET is then measured as the relative increase in the FRET channel over the expected background from spillover:
If there is no FRET then FRETR is 1. The maximum FRETR we have observed is approximately 2.5 for positive controls in which a protein is tagged with CFP and YFP in tandem using plasmid pDH18.