The first part of this series that provides a basic introduction to FRET and its applications can be read here.
The main player in a FRET experiment is the fluorescent donor molecule. This could be a small dye molecule, a Q-dot, or even a larger fluorescent protein such as GFP. The advantage of using fluorescent proteins such as GFP is that they can be genetically encoded to tag the protein of interest inside live cells. The second player is the acceptor molecule, which in most cases is a fluorophore itself. Choices of donor and acceptor should be based on the quantum efficiency of energy transfer between the pair, which depends on multiple factors, including spectral overlap. Unless there is sufficient overlap between the donor emission and acceptor excitation spectra, the two fluorophores will not transfer energy through FRET. There are plenty of FRET pairs available in literature, and some common ones are listed here.
Once you’ve determined the FRET pairs that you would like to use, it is important to clone the fluorescent tags to the proteins of interest at both the N and C termini to see which location causes the least effect on protein activity. If you do not have a readout of protein activity, try all combinations—donor N-tag + acceptor N-tag, donor C-tag + acceptor N-tag, donor N-tag + acceptor C-tag, and donor C-tag + acceptor C-tag to see which combination gives a good FRET signal.
Since random collision between donor and acceptor can also give you a FRET signal, negative controls are paramount to good FRET data. It is very common for people to use the acceptor fluorescent protein alone to measure the basal FRET with donor tagged protein and then subtract this signal from the FRET experiment results. However, the best control is to use a non-interacting protein of similar molecular weight or an interaction-defective mutant of your protein of interest. This will ensure that the diffusion kinetics of the negative control mirror the diffusion kinetics of the FRET experiment.
There are two other important parameters to measure before the actual FRET data collection can begin: cross excitation and bleed through. Cross excitation is the excitation of the acceptor fluorophore by the donor excitation beam. This will cause a background acceptor signal that is not due to FRET. Bleed through is the amount of light emitted by the excited donor in the acceptor wavelength region; due to the required spectral overlap between donor and acceptor, the contribution of donor bleed through to the acceptor emission needs to be subtracted from the FRET measurements as well. These two corrections are usually carried out by measuring in the FRET channel cells expressing donor tagged protein alone and cells expressing acceptor tagged protein alone.
Types of FRET measurements
The simplest way to measure FRET using a filter-based setup such as a fluorescence microscope or a multi-channel plate reader is sensitized emission (SE). This involves the creation of a separate FRET channel, which uses the excitation wavelength of the donor and collects emission at the wavelength of the acceptor. Once the individual tagged proteins are measured for cross-excitation and bleed through defects, the acceptor emission signal recorded in the FRET experiment is through the transfer of the energy to the acceptor by the excited donor. The signal coming from the negative control should also be measured through this FRET channel and subtracted from the data to give you the final values of FRET.
Another way to measure FRET is through acceptor photobleaching (AP). While the SE method uses the concept of measuring the excited acceptor, AP measures the gain in donor fluorescence when FRET between donor and acceptor is abolished. This method does not require a special FRET channel, nor does it require a fluorescent acceptor—although a fluorescent donor is necessary. At the start of the experiment, the intensity of the donor fluorescence is recorded using excitation and emission channels for the donor fluorophore. We can deduce that if FRET is taking place, only a part of the donor excitation light is used for donor emission, and the rest is being transferred to the acceptor.
The next step is to photobleach the acceptor. This can be done by exposing the sample to an acceptor excitation laser at high intensity, or to light of the excitation wavelength for a prolonged period of time. The loss of acceptor fluorescence through photobleaching can be measured by monitoring the acceptor excitation-acceptor emission channel. Once the acceptor is visibly bleached, the intensity in the donor excitation-donor emission channel is recorded again. Upon bleaching of the acceptor, FRET is lost and the excess energy present with the donor is emitted as donor fluorescence. An important control here is to measure the photobleaching of the donor alone under the same conditions.
Once you have acquired some FRET measurements, the last (and most enjoyable) phase is the data representation. SE recordings are usually represented as the ratio of the FRET channel (donor excitation – acceptor emission) to the donor channel (donor excitation – donor emission) on the Y-axis versus time on the X-axis. AP experiments can be shown as donor intensity versus time, with both pre-bleach and post-bleach recording on the same graph.
FRET is also completely amenable to visual representation; cells can be color coded according to SE intensities or donor intensities inside the cells before and after bleaching.
If you have access to a simple fluorescence microscope or a plate reader, any biomolecular interaction can be measured quickly and reliably using FRET. So let’s go out there and make FRET happen!
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