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.
- 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.
FRET is also ideally suited to High Throughput Screening (HTS) since it is simple, sensitive and easily automated.
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 – DyLight®650
- RPE – APC/Cy5.5
- Fluorescein – RPE
- APC – DyLight755
Click to read more
Reference: Prediction of breast cancer risk based on flow-variant analysis of circulating peripheral blood B cells – Genetics in Medicine (2017)
Download a Free Copy Lightning-Link® fluorescent antibody and protein labeling kits allow you to covalently label any antibody in under 20 minutes with only 30 seconds hands-on time. We currently have over 35 label in our range of fluorescent dyes. This guide provides a general overview of each fluorochrome in our range including their…View
Antibodies are widely employed in the quantification of antigens in complex biological samples. Using techniques such as Western blotting, ELISA, and immunohistochemistry researchers are able to measure a single antigen, or perhaps a limited number of antigens, in each sample. In the post-genomics era, advances in multiplex immunoassay technologies now allow scores or even hundreds…View
Expedeon specializes in easy to use bioconjugation kits which enable the direct labeling of antibodies or proteins with enzymes, fluorescent labels, biotin, streptavidin, gold nanoparticles, latex beads or oligonucleotides. Unfortunately, many antibodies are provided in buffers which contain additives that are incompatible with labeling technologies, making puri cation a key consideration prior to carrying out…View
Antibodies are used to detect and quantify antigens in techniques such as flow cytometry, ELISA, western blotting, immunohistochemistry and lateral flow. The antibody that binds to the antigen is called a ‘primary antibody’ and it confers specificity to the assay. Most types of immunoassays also incorporate a ‘label’ whose purpose is to provide measurability. Assays…View
Stop using secondary antibodies in immunoassays! Save time and materials by labeling your primary antibodies in under 20 min. For use in ELISA, western blotting, IHC, flow cytometry and many other applications. Scientists love our Lightning-Link® kits because of the remarkable simplicity of the technology. Now they can open a…View
**This webinar was produced by Innova Biosciences which is now fully integrated with their company, Expedeon Ltd., and have taken the Expedeon Ltd name and logo. Dr Nick Gee, the CEO and CSO of Expedeon, explains the benefits of using directly conjugated antibodies and the various approaches and techniques…View
**This webinar was produced by Innova Biosciences which is now fully integrated with our sister company, Expedeon Ltd., and have taken the Expedeon Ltd name and logo. FRET, or Fluorescence Resonance Energy Transfer, is a powerful technique dependent on the distance dependent transfer of energy from a donor molecule…View