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Biochemistry

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JoVE Journal Biochemistry
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

Article DOI: 10.3791/59656-v • 05:28 min • August 27th, 2019
August 27th, 2019 •
*1, *1, 1, 1, 1,2
1Department of Orthopaedic Surgery, New York University Medical Center, 2Department of Cell Biology, New York University School of Medicine
* These authors contributed equally

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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.

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Keywords: DARTS Drug Affinity Responsive Target Stability MTOR Rapamycin Protein-ligand Interaction Proteolytic Curve Dose-dependence Curve 293T Cells Cell Lysis BCA Assay Pronase Protease Inhibitor Cocktail SDS-PAGE
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