| Assay Method Information | |
| | Fluorescence Polarization Assay |
| Description: | Polarized fluorescence intensities were measured using an EnVision Multilabel plate reader (PerkinElmer) with excitation and emission wavelengths of 485 and 530 nm, respectively [Khanna et al., ACS Chem. Biol., 6:1232-1243]. A Thermo Scientific Nunc 384-well black microplate was used to prepare samples with a final volume of 50 μL in duplicate. First, the compounds were serially diluted in dimethyl sulfoxide (DMSO) and further diluted in 1× PBS buffer with 0.01% Triton X-100 to yield a final concentration from 100 to 0.046 μM. Triton X-100 was added in the buffer to avoid compound aggregation; 35 μL of the compound solution and 10 μL of PBS containing uPAR were added to the wells, and the mixture was incubated for at least 15 min to allow the compound to bind to the protein. Finally, 5 μL of fluorescent AE147-FAM peptide was added for a total volume of 50 μL in each well, resulting in final uPAR and peptide concentrations of 320 and 100 nM, respectively. |
| Affinity data for this assay | |
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