Real Experiences of uHTS: A Prototypic 1536-Well Fluorescence Anisotropy-Based uHTS Screen and Application of Well-Level Quality Control Procedures
| Autor: | Andrew J. Pope, Sandra Turconi, Stephen Ashman, David L. Earnshaw, Ryan P. Bingham, Murray J. B. Brown, Ulrich Haupts, Kenneth Fantom, Kerry Shea |
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| Rok vydání: | 2001 |
| Předmět: |
Boron Compounds
Quality Control 0301 basic medicine Reproducibility Binding Sites Chromatography Chemistry Analytical chemistry Reproducibility of Results Fluorescence Polarization 01 natural sciences Biochemistry Anti-Bacterial Agents 0104 chemical sciences Analytical Chemistry 010404 medicinal & biomolecular chemistry 03 medical and health sciences Autofluorescence 030104 developmental biology Escherichia coli Molecular Medicine Ribosomes Fluorescence anisotropy Fluorescent Dyes Biotechnology |
| Zdroj: | SLAS Discovery. 6:275-290 |
| ISSN: | 2472-5552 |
| Popis: | This paper describes, for the first time, a true ultra-high throughput screen (uHTS) based upon fluorescence anisotropy and performed entirely in 1536-well assay plates. The assay is based upon binding and displacement of a BODIPY-FL-labeled antibiotic to a specific binding site on 70S ribosomes from Escherichia coli (Kd approximately 15 nM). The screen was performed at uHTS rates (i.e.,100,000 assay wells/24 h) using entirely commercially available equipment. In order to examine the reproducibility of detection of test compound effects, assays were performed in duplicate. Both overall assay statistics and reproducibility for individual compound results were excellent, at least equivalent to conventional HTS assays. Interference artifacts occurred mainly as a result of autofluorescence from test compounds. Well-level quality control procedures were developed to detect, eliminate, or even correct for such effects. Moreover, development of a brighter, longer wavelength probe (based upon Cy3B) markedly reduced such interferences. Overall, the data demonstrate that fluorescence anisotropy-based uHTS is now a practical reality. |
| Databáze: | OpenAIRE |
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