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.
How HTRF technology assays work
The HTRF protocol is simple and straightforward. It requires few steps:
First add your analyte or sample to your microplate, then add the detection reagents: Anti-analyte conjugated donor and acceptor conjugated antibody. Incubate 2 hours (up to overnight for some assays). The assay is then ready to be read on an HTRF compatible or certified microplate reader. The emission of light by the acceptor will be proportional to the level of interaction.
Easy to implement
No washing required
Don’t do it alone, use a guide to get started!
PerkinElmer | Cisbio is actively contributing to research fields by creating new contents day after day, as well as more innovative products to help you get the most out of your experiments. These efficient key guidelines will take you further in your cell signaling, GPCR, cytokines, PPI, or Kinase experiments.