FRET SE is a method mainly used to analyze protein – protein interactions or conformational changes of proteins. There are several prerequisites for FRET to occur:
- The Donor and Acceptor distance must be <10 nm
- Sufficient separation of excitation and emission spectra are necessary
- The Donor emission spectrum must overlap with the Acceptor absorption spectrum
Due to the necessary overlap between Donor emission spectrum and Acceptor absorption spectrum each FRET measurement is done by sequential acquisition of three channels:
- Donor channel: Donor excitation and Donor emission
- FRET channel: Donor excitation, Acceptor emission
- Acceptor channel: Acceptor excitation and Acceptor emission
Donor and Acceptor channel are used to eliminate crosstalk into the FRET channel. FRET sample preparations must therefore include references of donor in the absence of the acceptor (donor only control) and acceptor in the absence of the donor (acceptor only control). Ideally, all references are included in the same preparation. The donor and acceptor references are used to obtain calibration coefficients to correct for excitation and emission cross talk.
As this method is non-invasive, it is most frequently used for live cell experiments.
It is important that throughout the entire experiment and calibration routine, all measuring parameters such as camera exposure time, gain, excitation intensities must remain constant.
