| Assay Method Information | |
| | Inhibition Assay |
| Description: | Materials: PAD1, PAD2, PAD3 and PAD4 were purchased from commercial sources. Assay buffer: 50 mM Tris pH 7.5, 100 mM NaCl, 10 mM CaCl2, 5 mM DTT. Color reagent: 1 volume of reagent A (80 mM diacetyl monoxime, 2 mM thiosemicarbazide) and 3 volumes of reagent B (17.35% v/v H3PO4, 33.7% v/v H2SO4, and 0.765 mg/mL ammonium iron (III) sulfate). The assays were conducted at 37° C. in the presence of each inhibitor in the concentration range of 1 to 1000 μM of each inhibitor tested. The inhibitor (12.5 μL of stock) was mixed with appropriate amount of substrate (BAEE) stock solution and preincubated at 37° C. for a period in the range of 0-60 min. The reaction was initiated by the addition of the enzyme. The reaction samples were incubated for a period in the range of 0-60 min. Color reagent was added (in the range of 50-300 μL/sample) and the samples were boiled for a period in the range of 0-45 min in a water bath. Samples were cooled on ice, vortexed and centrifuged. 200 μL aliquots were transferred to the 96-well plate and the absorbance was measured at 530 nm and the remaining activity was computed. |
| Affinity data for this assay | |
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