| Assay Method Information | |
| | Tumor Hsp90 Inhibitors Dose Response Confirmation |
| Description: | A fluorescence polarization based HTS assay has been developed and optimized for the identification of Hsp90 inhibitors by using tumor cell lysate Hsp90 and fluorescently (Cy3B) labeled geldanamycin (cy3B-GM) in 384-well black assay plate format. Fluorescence polarization in mP is measured at room temperature with an Analyst HT reader. An excitation filter at 545nm and an emission filter at 610 to 675nm are used with a dichroic mirror of 565nm. Assay data are analyzed using BioAssay software from CambridgeSoft. Percentage of inhibition is calculated by the equation based on per plate: % of Inhibition = 100 - ((mPc - mPf)/(mPb - mPf)) * 100 Where mPc is the recorded mP from compound wells; mPf is average recorded mP from cy3B-GM only wells; mPb is average recorded mP from wells containing cy3B-GM and NCI-N417 lysate. For each compound, a 4 parameter sigmoidal dose-response curve was fitted using BioAssay software from CambridgeSoft. The reported IC50 values were generated from fitted curves by solving for X-intercept at the 50% level of Y-intercept. When the highest concentration tested (50 micromolar) did not result in greater than 50% inhibition, the IC50 is reported as greater than 50 micromolar. Similarly, when the lowest concentration tested (1.5625 micromolar) resulted in greater than 50% inhibition, the IC50 is reported as less than 1.5625 micromolar. Compounds with IC50 values of greater than 30 micromolar were considered inactive. |
| Affinity data for this assay | |
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