| Assay Method Information | |
| | Fluorescence Polarization (FP Assay) |
| Description: | The ability of the compounds to compete with a fluorophore-labeled HIF-la peptide fragment (FITC-HIF-1α 788-822) for binding to the PHD2 protein was tested using a 384-well black plate (model: Corining #3575) with a final test volume of 60 μL. The compounds tested and FITC-HIF-1α 788-822 were dissolved in DMSO and purified water, respectively, for later use. The compounds were serially diluted in an assay buffer to 12 concentration gradients, and then 20 μL of diluted 300 nM FIH protein was added to each well. Two replicates were set for each compound concentration, and a blank control (20 μL FITC-HIF-1α 788-822+40 μL assay buffer) and a negative control (20 μL FITC-HIF-1α 788-822+20 μL FIH+20 μL assay buffer) were set for each assay. The plate was incubated at room temperature for 1 h and scanned with a Synergy plate reader. The excitation wavelength was set to 485 nm, and the emission wavelength was set to 535 nm. The calculation formula was as follows: % inhibition rate=100−[1−(measured−value blank)/(negative value−blank)], and the inhibition rate corresponding to a specific concentration was obtained. The obtained data were imported into Graphpad prism 8.0 for analysis and fit to obtain IC50 values. |
| Affinity data for this assay | |
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