BioAuxilium Thunder™ TR-FRET Cell Signaling Assays
BioAuxilium Thunder™ TR-FRET Cell Signaling Assays

What is FRET ? | What is TR-FRET ?

What is FRET?

Förster resonance energy transfer (FRET) describes a physical phenomenon of non-radiative energy transfer based on dipole-dipole coupling that can occur from an excited state fluorophore (the donor) to a ground state fluorophore (the acceptor) (Figure 1). FRET is detected by the appearance of sensitized fluorescence in the acceptor and by a decrease (quenching) of donor fluorescence.

Figure 1: FRET is based on the transfer of energy (not fluorescence!) between two suitable fluorophores, a donor and an acceptor. Excitation of the donor by a light source triggers a non-radiative energy transfer (FRET) towards the acceptor, which in turn emits sensitized fluorescence at a given wavelength.

FRET only occurs when the donor fluorescence emission spectrum overlaps with the acceptor excitation spectrum, when donor-acceptor relative dipole orientations are approximately parallel, and when the donor and acceptor are within close proximity (typically 1-10 nm). The latter has led to the widespread application of FRET in biomedical research for the study of molecular interactions, as well as the measurement of distances on a molecular scale.

Since the transfer of energy occurs only when the donor and acceptor are in close enough proximity, FRET based assays are homogeneous. That is, they do not require any washing or separation steps to remove distant donor and acceptor partners. However, the performance of traditional FRET assays using conventional fluorophores is often compromised by the susceptibility of conventional fluorophores to photobleaching, and by scattered light and high background fluorescence from sample components (e.g., cell debris, buffers, test compounds and microplates). This makes FRET signal-to-background (S/B) levels unsatisfactory for many applications.

What is TR-FRET?

Time-Resolved FRET (or TR-FRET) is a well-established technology widely used in drug discovery and clinical diagnostics, and increasingly used in academic laboratories. TR-FRET has turned out to be a highly versatile assay technology, allowing the study of a wide range of biological interactions ranging from low to high affinity, using both small and large molecules.

Figure 2: In contrast to standard FRET assays, THUNDER™ TR-FRET assays use a long-lifetime Europium chelate as the donor fluorophore. The specific FRET signal can thus be measured in a time-resolved manner, after a suitable time delay (typically 50 to 100 µs). This virtually eliminates all fluorescent background caused by the sample and plastic microplate, and by direct excitation of the acceptor. As a result, the THUNDER TR-FRET assays exhibit very low background and high S/B ratios.

TR FRET exploits the unique photophysical properties of lanthanide labels when used as FRET donors (Figure 2). Lanthanide labels have exceptionally long excited state lifetimes ranging from hundreds of microseconds to a few milliseconds. This is in sharp contrast to the very short fluorescent lifetime, on the low nanosecond range, of both conventional fluorophores used in standard FRET assays and natural background fluorescence. The long fluorescence decay after excitation allows time-resolved or gated FRET signal detection, after all interfering short-lived signals have decayed to negligible levels. The time-resolved detection also avoids interference from the acceptor emission due to its direct excitation (i.e., non-sensitized emission), rather than by FRET. In addition, lanthanide labels exhibit a large Stokes shift (the difference between the excitation and emission maxima) that minimizes crosstalk. Overall, because of their excellent temporal and spectral resolution, TR-FRET assays exhibit very low background and high S/B ratios, two features critical for the development of reproducible and robust assays. Of note, many microplate readers supporting TR-FRET measurements are available (see a list of TR-FRET compatible readers here).