Fluorescence-based detection techniques are popular in high throughput screening due to sensitivity and cost-effectiveness. Four commonly used techniques exist, each with distinct characteristics. Fluorescence intensity assays are the simplest to run, but suffer the most from signal interference. Fluorescence polarization assays show less interference from the compounds or the instrument, but require a design that results in change of fluorophore-containing moiety size and usually have narrow assay signal window. Fluorescence resonance energy transfer (FRET) is commonly used for detecting protein-protein interactions and is constrained not by the sizes of binding partners, but rather by the distance between fluorophores. Time-resolved fluorescence resonance energy transfer (TR-FRET), an advanced modification of FRET approach utilizes special fluorophores with long-lived fluorescence and earns its place near the top of fluorescent techniques list by its performance and robustness, characterized by larger assay window and minimized compound spectral interference. TR-FRET technology can be applied in biochemical or cell-based in vitro assays with ease. It is commonly used to detect modulation of protein-protein interactions and in detection of products of biochemical reactions and cellular activities.
TR-FRET Time-resolved fluorescence resonance energy transfer Protein-protein interaction Enzyme activity assay Product detection
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This work was supported by NIH Roadmap Initiatives grants U54HG003916 and U54HG005033. The protein-protein interactions protocol has been developed in a collaborative project with Dr. Yuan Chen (City of Hope). The enzymatic activity protocol has been developed in collaboration with Dr. Matt Petroski (Sanford-Burnham-Prebys MDI).
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