Time-Resolved Fluorescence Assays.
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.
Conrad Prebys Center for Chemical Genomics, Sanford-Burnham-Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA, 92037, USA.
Chen-Ting
CT
Ma
Conrad Prebys Center for Chemical Genomics, Sanford-Burnham-Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA, 92037, USA. esergien@sbpdiscovery.org.
Eduard A
EA
Sergienko
D000818:Animals; D044842:Cullin Proteins / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D000075687:NEDD8 Protein / Q000378:metabolism; D060066:Protein Interaction Maps / Q000187:drug effects; D025842:SUMO-1 Protein / Q000378:metabolism
D044842:Cullin Proteins; D000075687:NEDD8 Protein; D025842:SUMO-1 Protein
Enzyme activity assay; Product detection; Protein-protein interaction; TR-FRET; Time-resolved fluorescence resonance energy transfer