HTRF® (Homogenous Time-Resolved Fluorescence) is a Time-Resolved FRET technology developed by Cisbio Bioassays that can be conveniently performed using a microplate reader. It combines time-resolved methodologies with classical FRET to create assays of high robustness and sensitivity.
FRET (Fluorescence Resonance Energy Transfer) is based on the fact that a donor dye (e.g. CFP) in an excited state can transfer a part of its energy to an acceptor molecule like YFP. The emission from the acceptor can be detected as soon as both dyes are in close proximity when e.g. interaction of two proteins has taken place. Performance of traditional FRET assays is often hindered by background fluorescence from sample components, but this type of background fluorescence is extremely transient (with a lifetime in the nanosecond range) and can therefore be eliminated using time-resolved methodologies.
Time-Resolved Fluorescence (TRF) uses fluorophores with a very long fluorescence lifetime to avoid the interference caused by molecules with a short fluorescence lifetime or other factors (most importantly, excitation light). The fluorophores of choice are usually chelates of lanthanides (most commonly Europium, Terbium and Samarium).
In HTRF® the lanthanide chelate is used as donor and is either Europium cryptate (Eu3+ cryptate) or Lumi4™-Tb (Tb2+ cryptate), and the acceptor is either XL665, a modified allophycocyanin, or d2, a second generation of acceptor. In addition, HTRF® implements a ratiometric measurement that uses both the emission wavelength of the donor and of the acceptor to correct for well-to-well variability and signal quenching from assay components and media, that is absent in other TR-FRET assays.
HTRF® is a registered trademark of Cisbio Bioassays.