FRET

Fluorescence Resonance Energy Transfer (FRET) assays

Fluorescence occurs when a fluorophore moves to an excited state following the absorption of light at a specific wavelength, and subsequently produces a transient light emission at a higher wavelength as it returns to its ground state. The emitted light can be detected with a specialized reader.

Fluorescence Resonance Energy Transfer (FRET) is a distance-dependent interaction between two fluorophores. During FRET a donor fluorophore becomes excited by a light source, however it does not emit light following excitation but instead transfers its energy to an acceptor fluorophore. The acceptor fluorophore absorbs this and then produces a detectable light emission. This process results in the loss of fluorescence of the donor and gain of fluorescence of the acceptor, both of which can be measured.

For FRET to occur successfully, 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 = Ro6 / (Ro6 + r6), where Ro 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 Ro value usually lies between 10-100Å (1-10nm). FRET pairs with an Ro value towards the higher end of this range are often preferred due to the increased likelihood of FRET occurrence.
  • Spectral overlap. The emission spectrum of the donor fluorophore must overlap the absorption spectrum of the acceptor fluorophore. The greater the degree of spectral overlap, the more likely FRET is to occur.

    Successful FRET requires overlap between donor emission and acceptor absorbance spectra.

    Successful FRET requires overlap between donor emission and acceptor absorbance spectra, shown in grey.

  • 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 Ro.

FRET is an extremely powerful method of identifying molecular interactions, and can be applied within techniques such as flow cytometry, immunocytochemistry, immunohistochemistry and ELISA. One popular use of FRET is to identify an interaction between two biomolecules, for example the binding of a ligand to a receptor; a FRET signal will only be detectable when the biomolecules are in close proximity by virtue of a binding event.

The use of FRET to determine that an interaction between two biomolecules

The use of FRET to determine that an interaction between two biomolecules, for example ligand binding to a receptor, has taken place.

FRET is also ideally suited to High Throughput Screening (HTS) since it is simple, sensitive and easily automated.

FRET-based screening for inhibitors of kinase activity

FRET-based screening for inhibitors of kinase activity. Labeled antibodies against the target of a kinase, and its phosphorylated isoform,are brought in to close proximity following a phosphorylation event. Chemical inhibition of the kinase prevents phosphorylation and, since the antibody against the phosphorylated target will be lost during the wash steps of the assay, a FRET signal will not be detected.

FRET relies on the use of high quality labeled reagents. Depending on the intended assay setup these could be antibodies, proteins or peptides, however it is not feasible for the commercial market to supply all of these reagents prelabeled. Instead the end user may wish to label the reagents in-house.

Lightning-Link® antibody labeling kits

Lightning-Link® is an innovative technology that enables direct labeling of antibodies, proteins, peptides or any other biomolecule with free amine groups for use in a multitude of applications, including FRET. The kits offer several key advantages over traditional methods of conjugation:

  • Quick and easy to use
  • Require only 30 seconds hands-on time
  • No separation steps involved so 100% of the antibody or protein is recovered
  • Possibility to label from as little as 10ug to a gram or more of antibody

The Lightning-Link® range contains a wide range of fluorescent proteins and fluorescent dyes, and covers the spectrum from UV to far infrared. We also offer several tandem dyes, consisting of a donor fluorophore and an acceptor fluorophore which have been covalently attached to one another. Tandem dyes can be used to increase the quantity of readouts from a flow cytometry experiment, providing the opportunity for multiplexing.

We have identified the optimum FRET pairs when using our Lightning-Link® conjugation kits to be as follows:

  • RPE-APC
  • RPE-Cy5
  • RPE-Cy5.5
  • RPE-Cy7
  • RPE – DyLight®650
  • RPE – APC/Cy5.5
  • Fluorescein – RPE
  • APC – DyLight755


Reference
: Prediction of breast cancer risk based on flow-variant analysis of circulating peripheral blood B cells – Genetics in Medicine (2017)

To find out more about FRET, why not take a look at our webinar, or contact us directly.

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