How do you choose FRET pairs?

When choosing FRET pairs, choose the highest quantum yield donor, the highest absorbing acceptor, and fluorophores having substantial overlap in their spectral profiles. This combination will help maximize the FRET signal.

What are FRET pairs?

Fluorescence resonance energy transfer (FRET) is a distance-dependent interaction between the electronic excited states of two dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon.

How do you calculate r0 FRET?

In Fluorescence Resonance Energy Transfer (FRET) the FRET efficiency may be expressed as function of distances: E(r) = 1 / [1 + (r / R0)6], where r = distance between donor and acceptor chromophores, and R0 = Forster Radius with 50% Transfer Efficiency.

Which fluorescent protein might be used with GFP for doing a FRET experiment?

There are optimized FPs derived from the Aequorea GFP that are most often used for FRET-based imaging applications. Many early FRET studies relied on the Aequorea GFP derived blue FP (BFP), used in combination with GFP derivatives [5].

What is the principle of FRET?

The principle of FRET relies on the transfer of excitation energy of a donor fluorophore to a nearby acceptor fluorophore in a non-radiative fashion through long-range dipole-dipole interactions, when the distance separating them is 8 to 10 nanometers or less.

What is R0 FRET?

Page 1. Fluorescence Resonance Energy Transfer (FRET) Förster Radius. The distance at which energy transfer is 50% efficient (i.e. 50% of excited donors are deactivated by FRET) is defined by the Förster Radius (R0).

How do FRET sensors work?

The fundamental mechanism of FRET involves a donor fluorophore in an excited electronic state, which may transfer its excitation energy to a nearby acceptor fluorophore (or chromophore) in a non-radiative fashion through long-range dipole-dipole interactions.

What is the relationship between FRET efficiency and distance?

The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, making FRET extremely sensitive to small changes in distance.