TR-FRET

The measurement of tracer binding to the corresponding protein often relies on proximity. This induced proximity between the two moieties can be assessed by utilization of two fluorophores, which are attached to the protein and the tracer molecule. The Förster's Resonance Energy Transfer (FRET) between the two fluorophores is what allows for the spatial resolution. The emission wavelength of the one fluorophore excites the other fluorophore, which is not excited by the initial excitation wavelength. To increase the signal to noise ratio within FRET measurements, time-resolved FRET (TR-FRET) was developed for the elimination of short-lived background noise. This can be achieved by employing a lanthanide (Ln), typically terbium or europium, coupled with a short-lived (nanoseconds) fluorophore, which can be excited from the Ln. Since Ln conjugates display long-lived fluorescence (milliseconds), the Ln donor is excited and the acceptor signal is detected after a time-delay to avoid the short-lived fluorescence. Due to this, only tracers in close proximity to the tagged-protein of interest are detected.

For measurement, the Ln conjugates are linked to the protein of interest via e.g. an antibody-Ln conjugate or HaloTag-linked lanthanides. This allows in vitro measurements and in cellulo measurements in case of cell membrane permeable Ln cages.

TR-FRET can be a useful tool in a variety of applications including:

  • Live cell measurement:

    Payne NC, Kalyakina AS, Singh K, Tye MA, Mazitschek R.
    Bright and stable luminescent probes for target engagement profiling in live cells. Nat Chem Biol. 2021 Nov;17(11):1168-1177.

  • Functional studies of protein complexes:

    Adamson RJ, Payne NC, Bartual SG, Mazitschek R, Bullock AN.
    Structural and biochemical characterization establishes a detailed understanding of KEAP1-CUL3 complex assembly. Free Radic Biol Med. 2023 Aug 1;204:215-225.

  • Diagnostic use for detection of antibodies in patient samples:

    Yue, Hong, et al. Diagnostic TR-FRET assays for detection of antibodies in patient samples. Cell Rep Methods. 2023 Feb 20;3(3):100421.