TR-FRET Assays

Time-resolved fluorescence energy transfer (TR-FRET) is the practical combination of time-resolved fluorometry (TRF) combined 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.

The FRET aspect of the technology is driven by several factors, including spectral overlap and the proximity of the fluorophores involved, wherein energy transfer occurs only when the distance between the donor and the acceptor is small enough. Through measurement of this energy transfer, interactions between biomolecules can be assessed by coupling each partner with a fluorescent label and detecting the level of energy transfer. Acceptor emission as a measure of energy transfer can be detected without needing to separate bound from unbound assay components (e.g. a filtration or wash step) resulting in reduced assay time and cost.

Homogeneous, mix-and-read TR-FRET assays offer advantages over other biomolecular screening assays, such as fluorescence polarization (FP) or TRF assays. In FP assays, background fluorescence due to library compounds is normally depolarized and background signal due to scattered light (e.g. precipitated compounds) is normally polarized. Depending on the assay configuration, either case can lead to a false positive or false negative result. However, because the donor species used in a TR-FRET assay has a fluorescent lifetime that is many orders of magnitude longer than background fluorescence or scattered light, emission signal resulting from energy transfer can be measured after any interfering signal has completely decayed. TR-FRET assays can also be formatted to use limiting receptor and excess tracer concentrations (unlike FP assays), which can provide further cost savings. In the case of TRF assays, a wash step is required to remove unbound fluorescent reagents prior to measuring the activity signal of the assay. This increases reagent use, time to complete the assay, and limits the ability to miniaturize the system (e.g. converting from a 384-well microtiter plate to a 1536-well plate). TR-FRET assays take advantage of the required proximity of the donor and acceptor species for generation of signal.

FRET No Wash cAMP Assay

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Screen Quest™ FRET No Wash cAMP Assay Kit (Cat# 36379) provides a convenient assay method for monitoring the activation of adenylyl cyclase in G-protein coupled receptor systems. Compared with other commercial ELISA cAMP assay kits, this homogenous cAMP assay kit does not require a wash step or the acetylation step. The assay is based on the competition for a fixed number of anti-cAMP antibody binding sites between the fluorescent cAMP tracer and non-labeled free cAMP. Free cAMP displaces the fluorescent cAMP tracer from the HRP-cAMP/anti-cAMP antibody complex. The anti-cAMP antibody is labeled with our trFluor™ Eu while the cAMP tracer contains our cAMP-trFluor™ 650. In the absence of cAMP, cAMP-trFluor™ 650 conjugate is bound to trFluor™ Eu-labeled anti-cAMP antibody exclusively to have a strong FRET. However, the unlabeled free cAMP in the test sample competes for the trFluor™ Eu-labeled anti-cAMP antibody conjugate, therefore inhibiting the binding of cAMP-trFluor™ 650 to the anti-cAMP antibody. The cAMP-trFluor™ 650 labeled cAMP tracer only has a fluorescence lifetime in nanoseconds while trFluor™ Eu-labeled anti-cAMP antibody-bound fluorescent cAMP tracer has a much longer fluorescence lifetime value due to the TR-FRET. The magnitude of FRET is proportional to the concentration of cAMP in a sample.

trFluor™ Eu TRF Labeling Dyes

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AAT Bioquest's trFluor™ Eu probes enable TRF for the assays that require high sensitivity. The trFluor™ Eu dyes have large Stokes shifts and extremely long emission half-lives when compared with more traditional fluorophores such as Alexa Fluor^® or cyanine dyes. Compared with other time-resolved fluorescent probes, our trFluor™ Eu probes have relatively high stability, high emission yield and the ability to be linked to biomolecules with higher conjugation yields. Moreover, our trFluor™ Eu probes are insensitive to fluorescence quenching when conjugated to biological polymers such as antibodies. To maximize the TR-FRET potential, trFluor™ Eu dyes are optimized to pair with APC, iFluor® 647, TF5, Cy5^®, DyLight™ 650 and Alexa Fluor^® 647.

trFluor™ Tb TRF Labeling Dyes

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AAT Bioquest's trFluor™ Tb dyes have large Stokes shifts and extremely long emission half-lives when compared with more traditional fluorophores such as Alexa Fluor^® or cyanine dyes. Compared with other time-resolved fluorescent probes, our trFluor™ Tb probes have relatively high stability, high emission yield and ability to be linked to biomolecules with higher conjugation yield. Moreover, our trFluor™ Tb probes are insensitive to fluorescence quenching when conjugated to biological polymers such as antibodies. To maximize the TR-FRET potential, trFluor™ Tb dyes are optimized to pair with FITC, iFluor® 488, TF2, DyLight™ 488 and Alexa Fluor^® 488.