| Assay Method Information | |
| | Protease Inhibition Assays |
| Description: | The inhibition assays were performed in 96-well microtiter plates as follows: 30 uL of the enzyme solution (trypsin [100 ug/mL] or elastase [1.5 U/mL]) and 60 uL of the buffer (50 mM Tris-HCl, 20 mM CaCl2 at pH 7.5) were pipetted into a series of six wells. A total of 10 uL of a 2000 uM peptide solution (microviridin variant) was then added to well 1 and serially diluted up to well 5; well 6 was used as the control. After completing the serial dilution of the sample, 10 uL was taken from well 5 and discarded to maintain the same volume in all wells. The plates were then incubated for 5 min at 37 °C, and subsequently 90 uL of the substrate solution (trypsin assay, N-benzoyl-DL-arginine-4-nitroanilide hydrochloride (BAPNA) [2.17 mg mL-1]; elastase assay, succinyl-Ala-Ala-Ala-p-nitroanilide (Suc-ALA3-pNA) [1 mg mL-1]) was added to each well. The enzymatic activity was measured spectrophotometrically (Varioscan Flash, Thermo Scientific) at 410 nm after incubation at 37 °C for 15 m |
| Affinity data for this assay | |
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