Centre Optical Instrumentation Laboratory

University of Edinburgh

Supporting References

Periasamy and Day Methods Cell Biol. 1999;58:293-314.

Campbell et al PNAS, 2002 Jun 11;99(12):7877-82.

Erickson et al, Neuron 2001; 31: 973-985

Fluorescence Resonance Energy Transfer Protocol

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

(i) The Emission spectra of one fluorophore (Donor) must significantly overlap the excitation spectra of another fluorophore (Acceptor)
(ii) Donor and Acceptor must be aligned so that an acceptor dipole can be induced by the donor
(iii) The Donor/Acceptor separation can be no greater than 10nm
(iv) The Donor has a high quantum yield
(v) The Donor is saturated by the acceptor

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

E_T=	R₀⁶
	R⁶ + R₀⁶

R = Donor-Acceptor separation distance
R₀ = 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.

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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

Practical guides to FRET

Fluorescence Lifetime FRET

Acceptor photobleaching FRET

Sensitized Emission FRET