A comparison of ALPHAScreen, TR-FRET, and TRF as assay methods for FXR nuclear receptors
A comparison of ALPHAScreen, TR-FRET, and TRF as assay methods for FXR nuclear receptors
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DOI：
10.1177/108705710200700102
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发表时间：
2002-02-01
影响因子：
--
通讯作者：
Sills, M
中科院分区：
文献类型：
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作者：
Glickman, JF;Wu, X;Sills, M
检测技术的新发展提供了多种可供选择的生物分子筛选策略。因此，我们对基于分离和非基于分离的核受体配体筛选格式进行了详细分析。在本研究中，对时间分辨荧光共振能量转移（TR-FRET）、alphasgreen和时间分辨荧光（TRF）检测方法进行了优化，并在灵敏度、重现性和小型化能力方面进行了比较。结果表明，ALPHAScreen系统具有最佳的灵敏度和动态范围。由于必要的洗涤步骤的数量，TRF测定更耗时。TR-FRET测定的井间变化较小，很可能是由于比例测量。alphasgreen和TR-FRET试验都被缩小到8毫升体积。在光电倍增管阅读器中，alphasgreen阅读器（ALPHAQuest）通过同时读取四个光电倍增管，呈现出更快的读取时间的优势。
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-mul 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.