Time-Resolved FRET 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 proximity when the 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 like excitation light. The fluorophores of choice are usually chelates of lanthanides (most commonly Europium, Terbium and Samarium).
A powerful variant of TR-FRET is HTRF®.
Applications of TR-FRET
As an alternative to FRET, TR-FRET is often used to study protein-protein interactions. However, many other assays measuring the binding between two macromolecules have been developed.
Transcreener® GDP TR-FRET Red Assay with Mithras² Validation of the Mithras² Multimode Microplate Reader with the Transcreener® GDP TR-FRET Red assay
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Transcreener® GDP TR-FRET Red Assay with TriStar² S Validation of the TriStar² S Multimode Microplate Reader with the Transcreener® GDP TR-FRET Red assay
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cAMP/IP-One HTplex HTRF® assay with Mithras cAMP/IP-One HTplex cell-based experiment performed on Mithras LB 940 Multimode Microplate Reader using HTRF® technology
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Microplate Readers recommended for TR-FRET
A microplate reader capable of measuring high sensitivity fluorescence using filters and equipped with a flash lamp is the best combination for TRF (monochromator-based instrument may also be used but with lower performance). A detector with extended spectral range above 650 nm may be required for some applications. For example, some popular TR-FRET assays measure the Europium label at 665 nm.
HTRF® is a registered trademark of Cisbio Bioassays.