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Application of WST-8 based colorimetric NAD(P)H detection for quantitative dehydrogenase assays
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Document Title
Application of WST-8 based colorimetric NAD(P)H detection for quantitative dehydrogenase assays
Author
Chamchoy K., Pakotiprapha D., Pumirat P., Leartsakulpanich U., Boonyuen U.
Name from Authors Collection
Affiliations
Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, Bangkok, 10400, Thailand; Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand; Center for Excellence in Protein and Enzyme Technology, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand; Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok, 10400, Thailand; National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani, 12120, Thailand
Type
Article
Source Title
BMC Biochemistry
ISSN
14712091
Year
2019
Volume
20
Issue
1
Open Access
All Open Access, Hybrid Gold, Green
Publisher
BioMed Central Ltd.
DOI
10.1186/s12858-019-0108-1
Format
Abstract
Background: The reduction of tetrazolium salts by NAD(P)H to formazan product has been widely used to determine the metabolic activity of cells, and as an indicator of cell viability. However, the application of a WST-8 based assay for the quantitative measurement of dehydrogenase enzyme activity has not been described before. In this study, we reported the application of an assay based on the tetrazolium salt WST-8 for the quantitative measurement of dehydrogenase activity. The assay is performed in a microplate format, where a single endpoint is measured at 450 nm. Results: The optimized dehydrogenase-WST-8 assay conditions, the limit of detection (LOD), accuracy, and precision for measuring NAD(P)H, were demonstrated. The sensitivity of the WST-8 assay for detecting NAD(P)H was 5-fold greater than the spectrophotometric measurement of NAD(P)H absorption at 340 nm (LOD of 0.3 nmole vs 1.7 nmole, respectively). In the dehydrogenase assay, the colorimetric WST-8 method exhibits excellent assay reproducibility with a Z' factor of 0.9. The WST-8 assay was also used to determine dehydrogenase activity in biological samples, and for screening the substrate of uncharacterized short-chain dehydrogenase/oxidoreductase from Burkholderia pseudomallei. Conclusion: The results suggest that the WST-8 assay is a sensitive and rapid method for determining NAD(P)H concentration and dehydrogenase enzyme activity, which can be further applied for the high-throughput screening of dehydrogenases. © 2019 The Author(s).
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License
CC BY
Rights
Author
Publication Source
Scopus