FRET
Förster resonance energy transfer (FRET) 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 super super sensitive and lets 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 andbright 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 and acceptor fluorescent proteins, involves instrumental noise, 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 occurs through a chemical reaction which involves a substrate, some ions, molecular oxygen, and ATP. The good thing about BRET is that 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 is way easier compared to the meticulous one in FRET.
how do we utilize FRET?
First and foremost, we employ EGFP as a donor and mCherry fluorescent protein as an acceptor in our FRET experiments. Through various molecular cloning techniques, we tag our receptors, G proteins, or in general, our “protein of interest” with these two fluorescent proteins. The subsequent step involves inducing the expression of these fluorescently tagged proteins in cells and imaging them using our sophisticated laser scanning confocal microscope. In certain cases, we also measure the fluorescence using our “highly sensitive” plate reader.
and what about BRET?
Previously, we successfully utilized Renilla luciferase (hRLuc) in conjunction with DeepBlueC (coelenterazine 400a) as a donor and EGFP as an acceptor in our BRET setup. However, we have recently transitioned to the renowned NanoLuc protein, which exhibits significantly enhanced brightness when Furimazine is employed as a substrate and possesses a considerably smaller size compared to hRLuc. To achieve this, we simply tag the receptors or any protein of interest, followed by co-transfection of the cells to express the tagged proteins. Subsequently, we measure the BRET/bioluminescence using a plate reader.
