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A Semi-Quantitative Drug Affinity Responsive Target Stability (DARTS) assay for studying Rapamycin/mTOR interaction
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Biochimica
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JoVE Journal Biochimica
A Semi-Quantitative Drug Affinity Responsive Target Stability (DARTS) assay for studying Rapamycin/mTOR interaction

A Semi-Quantitative Drug Affinity Responsive Target Stability (DARTS) assay for studying Rapamycin/mTOR interaction

DOI:
10.3791/59656-v

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00:05 min

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August 27, 2019

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, , , ,
1Department of Orthopaedic Surgery,New York University Medical Center, 2Department of Cell Biology,New York University School of Medicine

Capitoli

  • 00:04Titolo
  • 00:47Collect and Lyse Cells
  • 01:59Incubate Protein Lysates with the Small Molecule and Perform Proteolysis
  • 03:28Western Blot Analysis
  • 03:59Results: Rapamycin Inhibition
  • 05:03Conclusion

Summary

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In this study, we enhanced the data analysis capabilities of the DARTS experiment by monitoring the changes in protein stability and estimating the affinity of protein-ligand interactions. The interactions can be plotted into two curves: a proteolytic curve and a dose-dependence curve. We have used mTOR-rapamycin interaction as an exemplary case.

Tags

DARTSDrug Affinity Responsive Target StabilityMTORRapamycinProtein-ligand InteractionProteolytic CurveDose-dependence Curve293T CellsCell LysisBCA AssayPronaseProtease Inhibitor CocktailSDS-PAGE

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