| Assay Method Information | |
| | Fluorescence Polarization Competition Assay |
| Description: | The assay was carried out in black half-area 96-well NBS plate (Corning), containing 15 nM of MCL-1 (BPS Bioscience), 5 nM of FITC-Bim and 3-fold serial diluted test compounds in a total volume of 50 μL of assay buffer (20 mM HEPES, 50 mM NaCl, 0.002% Tween 20, 1 mM TCEP, and 1% DMSO). The reaction plate was incubated for 1 hour at room temperature. The change of anisotropy is measured with an Envision multimode plate reader (PerkinElmer) at emission wavelength 535 nm. Fluorescence polarization was calculated in mP unit and the percentage inhibition was calculated by % inhibition=100×(mPDMSO−mP)/(mPDMSO−mPPC), in which mPDMSO is the DMSO control, and mPpc is the positive control. IC50 values were determined from a 10-point dose response curve by fitting the percent inhibition against compound concentration using the GraphPad Prism software |
| Affinity data for this assay | |
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