FRET - Image analysis - FRETR (Page 5)
FRETR is not a direct measure of the amount or efficiency of energy transfer.
FRETR is a relative and empirical measure of energy transfer. FRETR equals the
relative increase in the amount of light in the FRET channel when compared to
an expected baseline determined from the fluorescence in the YFP and CFP channels.
In order to best interpret FRETR one creates a positive control. The positive
control is constructed with pDH18 and creates a strain in which one of the
proteins of interest is fused to a tandem YFP-CFP tag. In practice we have
found that FRETR for our positive controls are typically 2.50 with a 10 -15%
standard deviation. If an experimental pair of proteins yields a FRETR of greater
than 2 than the proteins are in close proximity to each other. Of course, since
FRETR is an empircal measure biased in part by fluorescence filters, camera
specifications and the experimental set up, the value for the positive control
may be higher or lower.
What is the lower limit of detection? To push the sensitivity of FRETR one needs a
carefully chosen negative control. This would be a pair of proteins which a priori
one knows are too distant from each other (> 100 angstroms) to FRET. One can then
verify that the methodology and spillover factors return the expected FRETR value
of 1.00. In this case we have seen that FRETR values as low as 1.07 can represent
energy transfer. In the absence of negative controls certainly values of 1.20 and
higher represent energy transfer, assuming a positive control of 2.5.
This ends the tutorial on our approach to FRET in yeast. If you have found this tutorial helpful, or have further questions please register with our website. After registering click on the "Ask Question" tab under the bar. We need your input! Remember also that if you work on yeast we will do the FRET analysis. All you need to do is supply the strains.