| Assay Method Information | |
| | Fluorometric Assay |
| Description: | The capability of compounds described herein to inhibit the enzymatic activity of vascular adhesion protein-1 (VAP-1) was determined by fluorometric assay. The assay procedure provided by R&D Systems was modified slightly for use as an inhibitor screening assay. The assay measured the H2O2 generated from the oxidative deamination of benzylamine by rhVAP-1 by converting Amplex Red to the fluorescent product, resorufin, via horseradish peroxidase (HRP) and H2O2. Reaction buffer was 10 mM NaHCO3, pH 7.4. A standard curve was prepared by serially diluting H2O2 in reaction buffer. A 10 μM solution of resorufin was prepared in reaction buffer and was dispensed to 2 wells. These wells served as positive control for maximum fluorescence. Reaction buffer was added to the individual wells so that the final volume in each well was 100 μL. Amplex Red was diluted to 1 mM in reaction buffer. A 10 U stock of HRP was prepared in reaction buffer. A reaction cocktail was prepared by mixing equal parts 1 mM Amplex Red with 10 U HRP with 3 parts reaction buffers. The reaction cocktail was added to the appropriate wells, so that that final concentration in the well was 0.05 mM Amplex Red and 0.5 U HRP. A 200 μM solution of benzylamine was prepared in reaction buffer. Benzylamine was added to the appropriate wells so that the final concentration was 50 μM. Inhibitors were diluted in DMSO to 20 and distributed to the appropriate wells. UP-1207 served a positive control. rhVAP-1 was purchased from R&D Systems. The concentration used for each lot was determined so that the activity was equal to the first lot. Two wells were left blank as controls. The plate was incubated for 30 minutes at 37 C., protected from light under foil. The plate was read at 530/35 emission and 590/35 excitation. Data processing was performed using Excel and Prism software. |
| Affinity data for this assay | |
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