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?

2 probes:

  • 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.

Peter O'Toole, Cytometry Course 2019, York

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
Peter O'Toole, Cytometry Course 2019, York