A comparison of ALPHAScreen, TR-FRET, and TRF as assay methods for FXR nuclear receptors.
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-microl 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.
Glickman
J Fraser
JF
Novartis Institute for Biomedical Research, Summit, NJ 07901, USA. fraser.glickman@pharma.novartis.com
Wu
Xiang
X
Mercuri
Robert
R
Illy
Chantal
C
Bowen
Benjamin R
BR
He
Yang
Y
Sills
Matthew
M
D000818:Animals; D001331:Automation; D001647:Bile Acids and Salts / Q000378:metabolism; D001671:Biochemistry / Q000379:methods*; D004268:DNA-Binding Proteins / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D005982:Glutathione Transferase / Q000378:metabolism; D008024:Ligands; D008954:Models, Biological; D011485:Protein Binding; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors; D014157:Transcription Factors / Q000096:biosynthesis; D000099150:Receptor, Farnesoid X-Activated
D001647:Bile Acids and Salts; D004268:DNA-Binding Proteins; D005982:Glutathione Transferase; D008024:Ligands; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D000099150:Receptor, Farnesoid X-Activated