Fluorescence Resonance Energy Transfer

Fluorescence Resonance Energy Transfer Protocol

For Fluorescence Resonance Energy Transfer (FRET) to occur it must satisfy 5 criteria (Periasamy and Day 1999)

  1. The Emission spectra of one fluorophore (Donor) must significantly overlap the excitation spectra of another fluorophore (Acceptor).

  2. Donor and Acceptor must be aligned so that an acceptor dipole can be induced by the donor.

  3. The Donor/Acceptor separation can be no greater than 10nm.

  4. The Donor has a high quantum yield.

  5. The Donor is saturated by the acceptor.

Assuming that these criteria have all been met then energy transfer ET will occur and is described by the Forster equation.

R = Donor-Acceptor separation distance

Ro = Donor-Acceptor separation distance at which FRET efficiency is 50%


FRET Advantages/Disadvantages

Advantages

Disadvantages

Relatively cheap to implement

Only works if fluorophores are in correct orientation

Measurements are obtained very rapidly

Size of probes can introduce problems

Very good at measuring changes in distance

Gives no information about which probe moves

 

Free fluorophores can mask energy transfer

 

FRET pair labelling needs to be bright with the donor completely saturated by acceptor

 

Can be pH sensitive

 

If it doesn't work, it doesn't tell you anything, as you have no idea whether it failed due to incorrect orientation, free fluorophores etc or whether the proteins are simply too far apart.


There are generally 3 commonly used approaches for measuring FRET.

  • Decreased donor lifetime - measured using Fluorescence Lifetime Imaging Microscopy (FLIM) which requires expensive secondary equipment.

  • Decrease in Donor emission intensity - known as donor quenching, measured by acceptor photobleaching.

  • Increase in Acceptor emission intensity - known as sensitized emission.

fluorescence-lifetime-and-fluorescence-resonance-energy-transfer


Practical guides to FRET

Fluorescence Lifetime FRET

Acceptor Photobleaching FRET

Sensitised Emission FRET