An advantage of TR-FRET is the low background. Because of the long lifetime of the inorganic donor fluorescence, you can add a time delay between excitation and measurement. This allows the background fluorescence intensity from organic fluorophores to decay before the measurement. The resulting low background allows the donor concentration to be very low, and reduces interference from fluorescent compounds.

A disadvantage of TR-FRET is the extra cost of the equipment. I also think the short wavelength excitation is a disadvantage when screening compound collections because of inner filter effect by compound absorption of the excitation light.

Adam B Shapiro, thank you!

Dear Dr. Leonid A. Kaluzhskiy,

The difference in methods and usage of FRET and TR-FRET.

1. FRET:

• FRET is the radiationless transmission of energy from a donor molecule to an acceptor molecule.
• The donor molecule is the dye or chromophore that initially absorbs the energy and the acceptor is the chromophore to which the energy is subsequently transferred.
• FRET was first theorized and investigated by Theodor Förster in 1946. It was only in the 1970s that it was exploited in biologic and diagnostic systems.
• It is based on the transfer of energy between two fluorophores, a donor and an acceptor, when in close proximity.
• Molecular interactions between biomolecules can be assessed by coupling each partner with a fluorescent label and by detecting the level of energy transfer.
• Of course, FRET is governed by the physics of molecular proximity, which only allows this phenomenon to occur when the distance between the donor and the acceptor is short enough. In practice, FRET systems are characterized by the Förster’s radius (R0) distance at which FRET efficiency is 50%.
• The radius is highly dependant on the nature of fluorphores and partners.
• Because of these spectral properties, FRET, a donor-acceptor complex, can be detected without the need for physical separation from the unbound partners. Fully homogeneous assays do not require separation steps such as centrifuging, washing, filtration, or magnetic partitioning.
• These fluorophores can be coupled to interacting partners so that when the partners come close enough to each other, the dyes are mechanically brought into close proximity too (Angströms). Excitation of the donor by an energy source (e.g. a flash lamp or a laser) triggers an energy transfer towards the acceptor, which in turn emits specific fluorescence at a given wavelength.
• The donor and acceptor can be grafted covalently onto multiple partners that can associate, among others, two dimerizing proteins, two DNA strands, an antigen, and an antibody, or a ligand and its receptor.
• Traditional FRET chemistries are hampered by background fluorescence from sample components such as buffers, proteins, chemical compounds, and cell lysate.
• This type of background fluorescence is extremely transient (with a lifetime in the nanosecond range) and can, therefore, be eliminated using time-resolved methodologies.

2. TR-FRET:

• TR-FRET is a method that utilizes energy transfer measurements in the TRF instrument mode.  
• This allows homogeneous assay formats that can be read with high sensitivity.
• Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is a special form of resonance energy transfer (FRET) in which long emission fluorophores (lanthanides) are used as donors.
• The comparison measurement of the two emitted wavelengths over time is calculated for a TR-FRET response.
• TR-FRET) is the practical combination of time-resolved fluorometry (TRF) with Förster resonance energy transfer (FRET) that offers a powerful tool for drug discovery researchers.
• TR-FRET combines the low background aspect of TRF with the homogeneous assay format of FRET.
• The resulting assay provides an increase in flexibility, reliability, and sensitivity in addition to higher throughput and fewer false-positive/false-negative results. FRET involves two fluorophores, a donor and an acceptor.
• Excitation of the donor by an energy source (e.g. flash lamp or laser) produces an energy transfer to the acceptor if the two are within a given proximity to each other. The acceptor in turn emits light at its characteristic wavelength.
• Advantage: Homogeneous, mix-and-read TR-FRET assays offer advantages over other biomolecular screening assays, such as fluorescence polarization (FP) or TRF assays. One advantage of TR-FRET is that it places no particular size requirements on the binding pair, as does fluorescence polarization.
• Disadvantage: is that it requires two labels. A low dynamic range is a disadvantage.

I hope the information will help you.

With Best Wishes,

Samir G. Pandya

Samir G Pandya, thank you!

If you expect to have a significantly heterogeneous system, TR-FRET can help resolve the populations involved (given enough S:N to justify the model). Steady-state FRET (time-averaged FRET) will only give the average of the ensemble. This is important to protein folding investigations where you often have a distribution of distances; I don't see it's immediate application to binding.

I would suggest pressing your salesperson to tell you the value of TR-FRET. If you have doubts about the validity of their responses, bring them back here.

what does it mean when they tagged both fluorophores with the same antibody (Protein) for LANCE ULTRA TR-FRET detection assays ?

According to Perkin-Elmer, "LANCE Ultra uses a LANCE Europium chelate donor fluorophore and a ULight dye acceptor fluorophore." The principle is further described here:

https://www.perkinelmer.com/lab-products-and-services/application-support-knowledgebase/lance/lance-lance-ultra-tr-fret-assays.html

Ihab Abdallah if the donor and acceptor have the same antibody, then the experimenter expects both antibodies to come in close proximity to each other. I would expect that the antigen being studied potentially has two epitopes corresponding to the antibody used.