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| | Fluorescence Polarization Assay |
| Description: | An in vitro competition fluorescence polarization assay in which a test compound competes with a fluorescent probe for binding to the binding domain of human recombinant HSP90. The reaction can be followed kinetically using fluorescence (excitation lamda==485 nm; emission lamda==538 nm). The binding affinity of the test compound to HSP90 is determined by the changes in the polarized fluorescence; the intensity of the polarized fluorescence is proportional to the fraction of bound probe. To each test well, an aliquot of buffer, 2 ul of test compound in 10% DMSO, 4 ul of 6.25 nM of TSD FP probe, 4 ul of 12.5 nM of purified HSP90a protein were added. For positive control, 1 uM geldanamycin (GM) was used instead of the test compound (GM is a natural benzoquinone ansamycin that is known to bind to the N-terminal ATP-binding pocket of HSP90 and inhibits ATP binding and ATP-dependent chaperone activities). For negative control, no inhibitor was added. |
| Affinity data for this assay | |
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