Don’t FRET: Fluorescence Resonance Energy Transfer In a Nutshell
Fluorescence resonance energy transfer (FRET) is used to determine if two proteins are interacting in vivo. Each protein of interest is conjugated to a different fluorescent probe for detection. The absorption/excitation spectrum of one probe should partially coincide with the emission spectrum of the other. This allows for detection of all possible results: we will receive different fluorescent signals if the proteins interact, and if they do not. This increases confidence in our results.
Let’s consider a test case, wherein we are trying to determine if proteins A and B interact. We conjugate the fluorescent tag YFP (yellow fluorescent protein) to protein A, and CFP (cyan fluorescent protein) to protein B. These two are a good combination for FRET because the absorption spectrum of YFP partially overlaps with the emission spectrum of CFP.
Thus, when light in CFP’s absorption spectrum is shined on the system, CFP will emit light that excites YFP, which will emit yellow light that we can detect and interpret as a positive result of protein interaction. After all, if the fluorescent proteins are close enough to excite each other, it is very likely that the associated proteins (A and B) are interacting.
However, if proteins A and B do not interact, we will observe a different result. Upon shining a light in CFP’s absorption spectrum on the system, CFP will emit fluorescent light, as before. However, YFP will not be in the vicinity to be excited by CFP’s emission. Thus, we will observe cyan light, which in this case indicates a negative result.
In general, the fluorescent protein that emits light to the other is known as the donor protein, and the other is known as the recipient protein. Detecting the donor protein’s color indicates a negative FRET result, and detecting the recipient’s indicates the opposite.
Here are some additional sources if you want to learn more about FRET!
https://youtu.be/d2u5-CKBzSo - Watch this under-2-minute video summarizing pretty much everything I said.
https://youtu.be/1uPyq63aRvg - If you’ve been lost this whole time, my apologies. Watch this Bozeman Science video about absorption and emission spectra, and then maybe it’ll all make sense. Happy learning!
