| Assay Method Information | |
| | In Vitro Protease Assay |
| Description: | In vitro protease assays: Protease assays with IDE and neprilysin were performed using Mca-RPPGFSAFK(Dnp)-OH substrate peptide (R&D Systems, SEQ ID NO: 6) and purified recombinant human IDE and recombinant human neprilysin (R&D Systems) according to the manufacturer's instructions. For IDE, 2 μL of small molecule dissolved in DMSO was combined with 48 μL, 0.2 μg/mL IDE in IDE assay buffer (50 mM Tris, 1 M NaCl, pH 7.5). 50 μL, of 20 μM substrate peptide in IDE assay buffer was then added to initiate the reaction, and peptide cleavage was monitored on a fluorescent plate reader (excitation=320 nM; emission=405 nM) for 5 minutes. The above protocol was also used for assaying neprilysin activity, substituting the neprilysin assay buffer (50 mM Tris, 0.05% Brij-35, pH 9.0).Protease assays with THOP1 and NLN were performed using MCA-Pro-Leu-Gly-Pro-D-Lys(DNP)-OH (Bachem, SEQ ID NO: 7) and purified recombinant human THOP1 and NLN (R&D Systems) according to the manufacturer's instructions. |
| Affinity data for this assay | |
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