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A Comparison of ALPHAScreen, TR-FRET, and TRF as Assay Methods for FXR Nuclear Receptors
J. Fraser Glickman
Novartis Institute for Biomedical Research, Summit, NJ
Xiang Wu
Novartis Institute for Biomedical Research, Summit, NJ
Robert Mercuri
BioSignal-Packard, Montreal, Quebec, Canada
Chantal Illy
BioSignal-Packard, Montreal, Quebec, Canada
Benjamin R. Bowen
Novartis Institute for Biomedical Research, Summit, NJ
Yang He
Novartis Institute for Biomedical Research, Summit, NJ
Matthew Sills
Novartis Institute for Biomedical Research, Summit, NJ
New developments in detection technologies are providing a variety of biomolecular screening strategies from which to choose. Consequently, we performed a detailed analysis of both separation-based and non-separation-based formats for screening nuclear receptor ligands. In this study, time-resolved fluorescence resonance energy transfer (TR-FRET), ALPHAScreen, and time-resolved fluorescence (TRF) assays were optimized and compared with respect to sensitivity, reproducibility, and miniaturization capability. The results showed that the ALPHAScreen system had the best sensitivity and dynamic range. The TRF assay was more time consuming because of the number of wash steps necessary. The TR-FRET assay had less interwell variation, most likely because of ratiometric measurement. Both the ALPHAScreen and the TR-FRET assays were miniaturized to 8-µl volumes. Of the photomultiplier tube-based readers, the ALPHAScreen reader (ALPHAQuest) presented the advantage of faster reading times through simultaneous reading with four photomultiplier tubes.
Journal of Biomolecular Screening, Vol. 7, No. 1,
3-10 (2002)
DOI: 10.1177/108705710200700102
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