FRET
Förster resonance energy transfer is a non-radiative energy transfer through the dipole-dipole interactions between two fluorophores, which in our case are the fluorescent proteins. We love FRET because it is suuuuuper super sensitive and let us peek into live cells, image dynamic events such as protein-protein interactions with quite high spatiotemporal resolutions. If we delve a bit into the physics of FRET -which is kinda complicated and after a while might start to sound a little boring for some- the energy transfer is inversely proportional with the sixth power of the distance between the fluorophores AKA the donor and the acceptor. Furthermore, this sort of energy transfer is detectable only at the distances <10nm, making it highly specific and observable only if the proteins are truly interacting. These properties of FRET make it an ideal method for detecting inter- and intramolecular interactions especially in life sciences.
Although fluorescent proteins are quite handy in cell biology and allow us to detect multiple parameters in one well with numerous beautifully nice&bright color alternatives; yet one simply can’t look FRET through rose colored “optics” 🙂 When it comes to quantifying the actual FRET, the signal needs to be very well controlled. What we detect on the PMT also has bleed-throughs both from the donor & acceptor fluorescent proteins, involves instrumental noise etc etc etc. They all separately need to be calculated and pixel by pixel subtracted from the detected signal in the FRET channel.
what does it mean when there is a “B” instead of “F”?
The energy transfer theory also applies to BRET AKA “bioluminescence resonance energy transfer” with only one difference, in this case the donor is a bioluminescent protein and its excitation occur through a chemical reaction which involves a substrate, some ions, molecular oxygen and ATP. The good thing about BRET is it doesn’t need a light source hence we don’t need to worry about the bleed throughs. Its calculation needs a semi-straightforward ratiometric approach and way easier compared to the meticulous one in FRET.
how do we utilize FRET?
First of all, we are using EGFP as a donor and mCherry fluorescent protein as an acceptor in our FRET experiments. Using various molecular cloning methods, at the cDNA level we tag our receptors, G proteins or in general “our protein of interest” with these two fluorescent proteins. Next step is to make cells express these fluorescently tagged proteins and image them using our “fancy” laser scanning confocal microscope or in some cases measure the fluorescence with our “super sensitive” plate reader.
and what about BRET?
Previously, we successfully utilized Renilla luciferase (hRLuc) with DeepBlueC (coelenterazine 400a) as a donor and EGFP as an acceptor in our BRET setup. However, recently we switched to the famous NanoLuc protein which is far brighter when Furimazine is used as a substrate and way smaller compared to hRLuc. We simply tag the receptors or any protein we want to play with, then cotransfect the cells to express tagged proteins and measure the BRET/bioluminescence using the plate reader.
