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Assay Method Information |
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| | Fluorescence Polarization (FP) Assay |
| Description: | Assay buffer (25 μL, 20 mM HEPES at pH 7.3, 50 mM KCl, 5 mM MgCl2, 1 mM DTT, 20 mM Na2MoO4, 0.01% NP-40, and 0.5 mg mL−1 BGG) was added to a 96-well plate (black well, black bottom) followed by the desired compound at the indicated final concentrations in DMSO (1% DMSO final concentration). Subsequently, 10 nM recombinant cGrp94/Hsp90α and 6 nM FITC-GDA were added in 50 and 25 μL assay buffer respectively, resulting in a 100 μL final volume. Plates were incubatedfor 5 h at 4 °C on a rocker. Fluorescence was determined using excitation and emission filters of 485 and 528 nm, respectively. Percent FITC-GDA bound was determined by assigning the DMSO millipolarization unit (mP) value as the 100% bound value and 0% for FITC-GDA in assay buffer without any protein. |
| Affinity data for this assay | |
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