| Assay Method Information | |
| | Kinetics Assays |
| Description: | All assays were performed in TB supplemented with 0.2% Tween 20. For inhibition assays, recombinant DPP9 (25 nM) purified from insect cells was incubated with 2.5 uM recombinant SUMO1 for 2 h at 4 °C before adding different concentrations of the EIL peptide. DPP9 activity was then analyzed by measuring the hydrolysis of 250 uM H-Gly-Pro-7-amino-4-methylcoumarin (GP-AMC). For peptide screenings, recombinant DPP8 or DPP9 (25 nM) were incubated with 10 uM of each peptide and immediately tested for hydrolysis of 250 uM GP-AMC. For determination of Ki, varying concentrations of inhibitory peptides were added to recombinant enzymes purified from insect cells (12.5 or 25 nM) or immunopurified from HEK293T cells (12.5 nM) prior to assaying cleavage of GP-AMC (different concentrations) in the same buffer. In all cases, fluorescence release was measured using the Appliskan microplate fluorimeter (Thermo Scientific) with 380-nm (excitation) and 480-nm (emission) filters and SkanIt software and analyzed using Prism 5.0 (GraphPad software). Each experiment was performed at least three times, in triplicate. |
| Affinity data for this assay | |
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