| Assay Method Information | |
| | Assay for Inhibition of Thrombin by Compounds |
| Description: | For thrombin assay, a fluorescent peptide substrate (Boc-VPR-AMC (R&D Systems)) was used and reacted in a buffer containing 20 mM Hepes (pH 7.4), 140 mM NaCl, and 0.1% Tween 20 at room temperature. Assay parameters were adjusted to make the assay linearly related to time, enzyme, and substrate concentration. The assay method for thrombin is as follows. Test compounds were each serially diluted with a buffer, and then 10 μL of compound solution was transferred to an assay plate. A buffer was added to the assay plate at 10 μL/well, and then a thrombin (human α-thrombin (BioPharm Lab.)) solution was added at 15 μL/well. A Boc-VPR-AMC substrate solution was added at 15 μL/well, and the resulting mixture was incubated for 60 min at 25° C. A terminating solution (0.1 M acetic acid) was added at 15 μL/well, and detection was performed within 30 min by using a SpectraMax i3x microplate reader. |
| Affinity data for this assay | |
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