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HTRF Assay Principle
HTRF (Homogeneous Time Resolved Fluorescence) is a no-wash technology. It combines standard FRET technology with time-resolved measurement of fluorescence, eliminating short-lived background fluorescence. For a sandwich assay, two antibodies that recognize a protein of interest are used, with one antibody coupled to a donor, and the other with the acceptor. If the 2 antibodies recognize the analyte, the donor will emit fluorescence upon excitation and the energy will be transferred to the nearby acceptor, giving specific acceptor fluorescence. The donor fluorescence is also measured and a ratio with the acceptor fluorescence is applied.
HTRF®: the gold reference for time-resolved FRET
HTRF is a FRET-based technology that emerged from the Jean-Marie Lehn’s Nobel prize-winning work on rare earth complexes. Due to the chemical and photophysical characteristics of these fluorescent dyes, HTRF exhibits performance levels in stability and specificity that make it unparalleled for time-resolved FRET studies. It is especially extremely stable in time, allowing repetitive measurements for days. The very structure of HTRF reagents also makes assays highly resistant to most experimental conditions, including additives, chelates, DMSO, ionic strength, challenging cations, pH, temperature, or cell culture media. See the reagent chemistry tab for more details about HTRF reagents.