| Assay Method Information | |
| | Fluorometric PAI-1/tPA IC50 Plate Assay at pH 7.8 |
| Description: | PAI-1 inhibitor compounds were dissolved in DMSO to a final concentration of (10-50 mM), depending upon solubility. Compounds were then diluted in physiologic buffer (40 mM HEPES, 100 mM NaCl, 0.05% Tween-20, pH7.8) containing (10% DMSO and a dilution series (from 0 to 1000 μM depending on solubility) was prepared. 80 μL of compound was added per well to a 96-well black, opaque microplate in duplicate. 10 μL of 20 nM recombinant active human PAI-1 (Molecular Innovations) in physiologic buffer, as set out above, was added per well and the mixture was agitated for 15 minutes at room temperature. 10 uL of 25 nM human tissue type PA (tPA) (Activase (alteplase), Genentech) was added per well and the plate was agitated for an additional 30 minutes at room temperature. Tissue type PA activity in each reaction mixture was determined by adding Phe-Gly-Arg-AMC fluorogenic substrate (100 μL of 100 μM) (Centerchem). The rate of AMC release by tPA was measured at an excitation wavelength of 370 nm and an emission wavelength of 440 nm. Controls included PAI-1 and tPA in the absence of compound and tPA alone. Percent PAI-1 inhibition was calculated using the following formula: [(tPA alone-tPA/PAI-1+ compound)/(tPA alone-PAI-1/tPA)]*100%. The IC50 is calculated using Graphit (IC50 0-100%). |
| Affinity data for this assay | |
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