FRET: Fluorescence Resonance Energy Transfer
What it does best:
Enables investigation of molecular interactions. It depends on the transfer of energy from one fluorophore to another fluorophore when two molecules are in close proximity. Process by which radiationless transfer of energy occurs from an excited state fluorophore to a second chromophore in close proximity. Because the range over which the energy transfer can take place is limited to approximately 10 nanometers, and the efficiency of transfer is extremely sensitive to the separation distance between fluorophores, resonance energy transfer measurements can be a valuable tool for probing molecular interactions. Permits determination of the approach between two molecules within several nanometers, a distance sufficiently close for molecular interactions to occur.
What it can’t do:
Examples of uses:
Protein interactions. Almost any molecule can be labeled for FRET studies. Valuable tool for investigating and quantifying interactions in signal transduction, the formation of protein complexes, protein-protein interactions receptor function, membrane dynamics and molecular spatial relationships.
General hints and tips:
Following conditions must be satisfied for FRET:
- The donor probe should have sufficiently long lifetime for energy transfer to occur
- Donor and acceptor molecules must be approximately 1-10nm apart
- The absorption spectrum of the acceptor fluorophore must overlap the fluorescence emission spectrum of the donor fluorophore (by approximately 30%)
- For energy transfer, the donor and acceptor dipole orientations must be approximately parallel
Energy transfer is demonstrated by quenching of donor fluorescence together with a reduction in the fluorescence lifetime, and an increase in acceptor fluorescence emission. FRET is very sensitive to the distance between fluorophores and can be used to estimate intermolecular distances. FLIM imaging can be used in association with FRET studies to identify and characterize energy transfer.