Fluorescence Polarization Assay for HSP90α Inhibitor Binding

Competitive binding of inhibitors to HSP90α was monitored using FITC-GM in a FP assay [47 (link)]. Briefly, compound dilutions were incubated with HSP90α (90 nM final) and FITC-GM (2 nM final) in a 96-well microplate at 4°C for 16 h in the presence of assay buffer (20 mM HEPES, pH 7.4, 50 mM KCl, 5 mM MgCl₂, 20 mM NaMoO₄, 0.01% NP40, 2 mM DTT and 0.1 mg/ml BSA). Polarization was measured in Synergy H4 Hybrid reader (BioTek, Winooski, VT, USA) using Gen5.0 software (BioTek). [49 (link)]

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Zhao Z., Zhu J., Quan H., Wang G., Li B., Zhu W., Xie C, & Lou L. (2016). X66, a novel N-terminal heat shock protein 90 inhibitor, exerts antitumor effects without induction of heat shock response. Oncotarget, 7(20), 29648-29663.

Publication 2016

Synergy H4 Hybrid reader Gen5.0 software

Assay Buffer Dilutions Fitc Hepes Hybrid Inhibitors Mgcl2

Corresponding Organization :

Other organizations : Shanghai Institute of Materia Medica, Chinese Academy of Sciences

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Variable analysis

independent variables

Compound dilutions

dependent variables

Polarization

control variables

HSP90α (90 nM final)
FITC-GM (2 nM final)
Assay buffer (20 mM HEPES, pH 7.4, 50 mM KCl, 5 mM MgCl2, 20 mM NaMoO4, 0.01% NP40, 2 mM DTT and 0.1 mg/ml BSA)
Temperature (4°C)
Incubation time (16 h)

positive controls

No information provided

negative controls

No information provided

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