Fluorescence resonance energy transfer

During fluorescence resonance energy transfer (FRET), a donor fluorophore is excited by a light source and transfers its energy to a nearby acceptor fluorophore. The acceptor fluorophore absorbs the energy to produce a detectable light emission signal. This process results in the donor's fluorescence loss and the acceptor's fluorescence gain, both of which can be measured.​

FRET means we can detect when two molecules are within several nanometers of each other. This distance-dependent interaction provides information on potential interactions between labeled molecules within a cell.

As a result, FRET is a powerful method for identifying potential molecular interactions and can be used in techniques such as flow cytometry, immunocytochemistry, immunohistochemistry, and ELISA. FRET is also ideally suited to high-throughput screening (HTS) since it is simple, sensitive, and easily automated. Another popular use of FRET is to identify an interaction between two biomolecules, such as the binding of a ligand to a receptor.

​​For FRET to occur, several conditions must be met:

• Proximity: The donor and acceptor fluorophores must be close to one another for the FRET process to be efficient. 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 two fluorophores. 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 (1-10 nm). FRET pairs with a R₀ value towards the higher end of this range are often preferred due to the increased likelihood of FRET occurrence.

• ​​Spectral overlap: The donor fluorophore's emission spectrum must overlap the acceptor fluorophore's absorption spectrum. The greater the degree of spectral overlap, the more likely FRET is to occur.

• ​​​Dipole orientation: Energy transfer from the donor fluorophore to the acceptor fluorophore occurs via intermolecular dipole-dipole coupling. The spatial relationship between the donor emission dipole moment and the acceptor absorbance dipole moment is described by the orientation factor, ĸ2, which has values ranging from 0 (all dipoles are perpendicular) to 4 (all dipoles are parallel). Dipole orientation between the two fluorophores is typically assumed to be random due to rapid molecular rotation and is taken to equal the statistical average (a value of 2/3) for most calculations of R₀.

Optimal fluorophore pairs for FRET

FRET relies on the use of high-quality labeled reagents. Depending on the intended assay setup, these could be antibodies, proteins, or peptides. The following fluorophore pairs have been identified as optimal to use in FRET:

• ​RPE-APC
• RPE-Cy5
• RPE-Cy5.5
• RPE-Cy7
• RPE – DyLight®650
• RPE – APC/Cy5.5
• APC – DyLight750

Find out more about Abcam's labeled reagents for FRET on our website.