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A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors
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Biochemistry
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JoVE Journal Biochemistry
A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

DOI:
10.3791/60017-v

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10:28 min

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August 17, 2019

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, , , ,
1Department of Molecular Medicine,The Scripps Research Institute, 2Department of Neuroscience,The Scripps Research Institute, 3Laboratory of Molecular Physiology, NHLBI,National Institutes of Health

Chapters

  • 00:04Title
  • 00:46Measuring ATPase Activities and Inhibitory Effects of Small Molecule Inhibitors
  • 05:48Analyzing Data
  • 07:54Results: Fluorescence Intensity
  • 09:26Conclusion

Summary

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A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 µL per well. The platform should be applicable to virtually any ADP producing enzyme.

Tags

Keywords: ATPase AssayNADH-coupledSemi-high-throughput ScreeningActinMyosin IIEnzyme InhibitorsCalibrationPositive And Negative Controls

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