AAT Bioquest

Förster Resonance Energy Transfer (FRET)

Fluorescence resonance energy transfer (FRET) is a distance-dependent transfer of energy between two chromophores, a donor and an acceptor molecule. When in close proximity, electronically excited donor chromophores can transfer energy nonradiatively to acceptor chromophores through intramolecular long-range dipole-dipole coupling interactions. As a result, the transfer of energy quenches the fluorescence intensity of the donor and reduces its excited-state lifetime while increasing the emission intensity of the acceptor chromophore. Since the efficiency of FRET is dependent on the inverse sixth power of intermolecular separation, it is an advantageous technique for investigating various biological phenomena that produce changes in molecular proximity, including receptor-ligand interactions, spatial distribution, and assembly of protein complexes, and membrane potential sensing.

AAT Bioquest offers a large assortment of fluorescent donor and acceptor pairs and non-fluorescent Tide Quencher™ and BXQ dyes for engineering FRET-based biosensors, as well as FRET peptides for investigating coronavirus protease activity and no-wash, time-resolved FRET assays for monitoring the activation of adenylyl cyclase in G-protein coupled receptor systems.



Three Primary Conditions for FRET

FRET - spectral overlap
Schematic representation of the FRET spectral overlap integral (shown in gray shadow).
While many factors can influence FRET efficiency, three primary conditions must be satisfied for FRET to occur.
  1. The donor and acceptor molecules must be in close proximity to one another, typically 10-100 Å (1-10 nm). FRET efficiency (E) is defined by the equation E = R0⁶/(R0⁶ + r⁶), where R0 is the Förster radius, and r is the actual distance between the donor and acceptor molecules. The Förster radius is the distance at which 50% of the excitation energy is transferred from the donor to the acceptor, and the R0 value usually lies between 10-100 Å. FRET pairs with an R0 value towards the higher end of this range are often preferred due to the increased likelihood of FRET occurrence.
  2. The absorption or excitation spectrum of the acceptor must overlap the fluorescence emission spectrum of the donor (Figure 1). The degree to which they overlap is referred to as the spectral overlap integral (Jλ, grey shaded region). The greater the degree of Jλ, the greater the likelihood FRET is to occur.
  3. The donor and acceptor transition dipole orientations must be approximately parallel.

Spectral Overlap (Jλ) = ∫FD(λ)&εA(λ)λ⁴d(λ)
FD(λ)Is the corrected fluorescence intensity of the donor in the wavelength range λ to λ+∆λ with the total intensity (area under the curve) normalized to unity
εAIs the extinction coefficient of the acceptor at λ in units of M-1cm-1
λIs the wavelength in nm


Selecting Donor/Acceptor FRET Pairs

Selecting the appropriate donor and acceptor chromophores pair is essential for improving FRET efficiency. As aforementioned, optimal FRET pairs should satisfy these specifications:
  1. Select FRET pairs that maximize the spectral overlap of donor emission and acceptor excitation.
  2. Donor and acceptor pairs must have a sufficient spectral difference such that they are distinguishable from each other.
  3. Acceptor fluorophores should have minimal direct excitation at the absorbance maximum of the donor fluorophore to minimize any potential cross-talk.
Additional parameters such as donor quantum yield and the extinction coefficient of the acceptor can also affect FRET efficiency. Therefore, select donor and acceptor chromophores that are complementary to one another. For instance, selecting a donor with the highest quantum yield and pairing it with the highest absorbing acceptor, assuming sufficient spectral overlap between the pair, will maximize the FRET signal. In situations of low FRET efficiency, a more efficient FRET pair will significantly improve the detectable FRET interaction.

Donor-acceptor pairs may consist of the same or different chromophores. In most applications, the donor and acceptor chromophores are different. In these scenarios, FRET is identified by the presence of sensitized fluorescence of the acceptor chromophore or the fluorescence quenching of the donor chromophore. The latter method is independent of the acceptor chromophores' fluorescence capabilities. If the acceptor is fluorescent, FRET can be also be detected by the intensity ratio change of the donor/acceptor.

Several R0 values for various donor-acceptor chromophore pairs have been identified and published. FRET pairs with larger R0 values are indicative of higher FRET efficiency. A commonly used chromophore pair for developing FRET-based assays is fluorescein and tetramethylrhodamine. Together this FRET pair has an R0 value ranging between 49-56 Å R0 values of common donor-acceptor pairs are listed in Table 1. For a more extensive assortment of AAT Bioquest's Tide Fluor™ (TF) and Tide Quencher™ (TQ) FRET pair, refer to Appendix A. Our recommended TR-TQ FRET pairs demonstrate superior sensitivity and low background interference in protease and nucleic acid detection assays.

Table 1. Common FRET donor and acceptor pairs and their R0 values.

IAEDANS*5-IAF (5-Iodoacetamidofluorescein)49


Ordering Information


Table 2. Ordering Information For All FRET Building Blocks

Table 3. trFluor™ Products

Product Name
Unit Size
1300ReadiLink™ Rapid trFluor™ Eu Antibody Labeling Kit *Microscale Optimized for Labeling 50 ug Antibody Per Reaction*2 Labelings
1302Buccutite™ Rapid trFluor™ D2 Acceptor Antibody Labeling Kit *Microscale Optimized for Labeling 100 ug Antibody Per Reaction*2 Labelings
1305ReadiLink™ Rapid trFluor™ Tb Antibody Labeling Kit *Microscale Optimized for Labeling 50 ug Antibody Per Reaction*2 Labelings
1430trFluor™ Eu-Cryptate succinimidyl ester100 ug
1431trFluor™ Eu-Cryptate succinimidyl ester1 mg
1433trFluor™ Eu succinimidyl ester1 mg
1434trFluor™ Eu maleimide100 ug
1440trFluor™ Eu Acceptor XL6651 mg
1443trFluor™ Tb succinimidyl ester1 mg
1444trFluor™ Tb maleimide100 ug
1446trFluor™ Eu DBCO100 ug
16518trFluor™ Eu goat anti-mouse IgG (H+L)100 ug
16755trFluor™ Eu goat anti-mouse IgG (H+L)1 mg
16519trFluor™ Tb goat anti-mouse IgG (H+L)100 ug
16756trFluor™ Tb goat anti-mouse IgG (H+L)1 mg
16598trFluor™ Eu goat anti-mouse IgG (H+L) *Cross Adsorbed*100 ug
16791trFluor™ Eu goat anti-mouse IgG (H+L) *Cross Adsorbed*1 mg
16599trFluor™ Tb goat anti-mouse IgG (H+L) *Cross Adsorbed*100 ug
16792trFluor™ Tb goat anti-mouse IgG (H+L) *Cross Adsorbed*1 mg
16668trFluor™ Eu goat anti-rabbit IgG (H+L)100 ug
16820trFluor™ Eu goat anti-rabbit IgG (H+L)1 mg
16669trFluor™ Tb goat anti-rabbit IgG (H+L)100 ug
16821trFluor™ Tb goat anti-rabbit IgG (H+L)1 mg
16725trFluor™ Eu goat anti-rabbit IgG (H+L) *Cross Adsorbed*100 ug
16847trFluor™ Eu goat anti-rabbit IgG (H+L) *Cross Adsorbed*1 mg
16726trFluor™ Tb goat anti-rabbit IgG (H+L) *Cross Adsorbed*100 ug
16848trFluor™ Tb goat anti-rabbit IgG (H+L) *Cross Adsorbed*1 mg
16925trFluor™ Eu-streptavidin conjugate100 ug
16926trFluor™ Tb-streptavidin conjugate100 ug
62000trFluor™ Eu donkey anti-goat IgG (H+L) *Cross Adsorbed*50 ug
62005trFluor™ Eu donkey anti-rat IgG (H+L) *Cross Adsorbed*50 ug

Table 4. Ordering Information for AzoDyes