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Journal of Biomolecular Screening
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Utilization of Substrate-Induced Quenching for Screening Targets Promoting NADH and NADPH Consumption

María Jesús Vázquez

Assay Development, Glaxo SmithKline, Madrid, Spain

Stephen Ashman

Assay Development, Glaxo Smith Kline, Essex, United Kingdom

Fernando Ramón

Screening and Compound Profiling, Glaxo Smith Kline, Madrid, Spain

David Calvo

Assay Development, Glaxo Smith Kline, Madrid, Spain

Ana Bardera

J. Julio Martín

Screening and Compound Profiling, Glaxo Smith Kline, Madrid, Spain

Martin Rüdiger

David Tew

Assay Development, Glaxo Smith Kline, Essex, United Kingdom

Juan Manuel Domínguez

Assay Development, Glaxo Smith Kline, Madrid, Spain

Oxidation of reduced nicotinamide adenine dinucleotides is a common event for many biochemical reactions. However, its exploitation for ultrahigh-throughput screening purposes is not an easy task and is affected by various drawbacks. It is known that such nucleotides induce quenching on the fluorescence of several dyes and that this quenching disappearswith oxidation of the nucleotide. We have made use of this property to develop an assay for high-throughput screening with NADH and NADPH-dependent reductases. Full screening campaigns have been run with excellent assay quality parameters, and interesting hits have been identified. The method is amenable to miniaturization and allows easy identification of false positives without needing extra secondary assays. Although it is based onmonitoring substrate consumption, it is demonstrated that the effect of fractional conversion on assay sensitivity is negligible.

Key Words: substrate-induced quenching • high-throughput screening • NADH • NADPH • dehydrogenase • quenching

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This version was published on February 1, 2006

Journal of Biomolecular Screening, Vol. 11, No. 1, 75-81 (2006)
DOI: 10.1177/1087057105283296
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This Article
Right arrow Abstract Freely available
Right arrow Free Full Text (Free PDF) Free
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to Saved Citations
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Request Reprints
Right arrow Add to My Marked Citations
Citing Articles
Right arrow Citing Articles via Web of Science (1)
Right arrow Citing Articles via Google Scholar
Right arrow Citing Articles via Scopus
Google Scholar
Right arrow Articles by Vázquez, M. J.
Right arrow Articles by Domínguez, J. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vázquez, M. J.
Right arrow Articles by Domínguez, J. M.
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What's this?