CHEMBL5734553

Fluorescence Polarization Assay: Fluorescence polarization assay was performed to confirm the binding ability to the compound synthesized with HSP90 according the concentration. 1 M dithiothreitol (DTT), 10 mg/m: BGG (bovine gamma globulin) were added to hexafluorobenzene (HFB) buffer (20 mM HEPES pH 7.3, 50 mM KCl, 5 mM MgCl2, 20 mM Na2MoO4, 0.01% NP4O, 3rd distilled water) and mixed. Then, 100 nM gambogic acid (GA)-FITC (Fluorescein isothiocyanate) was added and reacted at room temperature for 10 minutes. Each 100 μL of the mixture was dispensed into the control wells of a 96-well plate, 2 μL of HSP90α was added to the remaining mixture, and each 100 μL of the other wells was dispensed. Each 98 μL was dispensed into the wells to which the compound was to be treated, and then each 2 μL was added at 5 mM, 2.5 mM, 500 μM, 50 μM, 25 μM, 5 μM, 500 nM or 50 nM for each concentration. After 1, 4, 6, or 18 hours of reaction, fluorescence polarization was measured at 495/530 nm by a plate reader.