| Assay Method Information | |
| | AlphaScreen Competitive Assay |
| Description: | HSP90-binding activity was determined by an AlphaScreen competitive assay system. The purified HSP90 solution was diluted with a binding buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Triton-X 100, 1 mM DTT, 0.1% BSA) and added to a 384-well plate (#3673, Corning Inc.) containing test substances. After reaction at room temperature for 2 hours, biotinylated geldanamycin was added thereto at a concentration of 40 nM/well and further reacted for 1 hour. Detection mix (20 mM HEPES-KOH (pH 7.5), 0.5% BSA, 0.04 mg/mL Nickel Chelate Acceptor beads, 0.04 mg/mL Streptavidin-coated Donor beads) (#6760619C, PerkinElmer, Inc.) was added to each well in an amount equal to the amount of the reaction solution and reacted at room temperature for 1 hour in the dark. Then, fluorescence intensity was measured using Multilabel Plate Reader EnVision (PerkinElmer, Inc.). |
| Affinity data for this assay | |
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