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Nano-Differential Scanning Fluorimetry for Screening in Fragment-based Lead Discovery
JoVE 杂志
生物化学
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JoVE 杂志 生物化学
Nano-Differential Scanning Fluorimetry for Screening in Fragment-based Lead Discovery

Nano-Differential Scanning Fluorimetry for Screening in Fragment-based Lead Discovery

DOI:
10.3791/62469-v

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06:26 min

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May 16, 2021

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, , , , , , , ,
1Oncode Institute and Division of Biochemistry,the Netherlands Cancer Institute, 2Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance (BMRZ),Johann Wolfgang Goethe-University

Chapters

  • 00:04Introduction
  • 00:35Plate Preparation
  • 01:06Fragment Nano-Dispensing by the Mosquito Robot
  • 02:01Fragment Dispensing
  • 03:17Preparing the Prometheus
  • 04:25Fluorescence Scan and Thermal Denaturation
  • 05:04Results: Fragment Screening
  • 05:49Conclusion

Summary

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Monitoring changes in the melting temperature of a target protein (i.e., thermal shift assay, TSA) is an efficient method for screening fragment libraries of a few hundred compounds. We present a TSA protocol implementing robotics-assisted nano-Differential Scanning Fluorimetry (nano-DSF) for monitoring intrinsic tryptophan fluorescence and light back-scattering for fragment screening.

Tags

Keywords: Nano-differential Scanning FluorimetryFragment-based Lead DiscoveryProtein SampleLigand ScreeningFragment PlateProtein PlateNano DispenserDeck ConfigurationFragment DispensingProtein Sample Preparation

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