FRET - Fluorescence Resonance Energy Transfer
- method for assessment of protein-protein interaction using fluorescent tags, eg. CFP and YFP;
- FRET proves if molecules are within 10nm or closer e.g. heterodimers, DNA bound molecules;
- accuracy of visual co-localisation with light microscopy is ~500nm, so molecules may not really be together;
What probes are required for cytometry based FRET?
- Donor molecule, which is excited
- Acceptor molecule, which emits
Donor emission spectrum has to overlap acceptor excitation spectrum.
Energy passed on in a non-radiative manner.
FRET by sensitised emission
1 Excite Donor control (e.g. CFP) - Compensate signal from FRET channel
2, 3 Excite Acceptor control (e.g. YFP) with 405 and 488 nm lines - Compensate YFP signal from FRET channel
4 Run experimental sample
- Donor excited - energy passed onto acceptor (e.g. YFP)
- Acceptor emits photon into FRET channel, donor fluorescence is reduced