| Assay Method Information | |
| | DPP1 Enzyme Activity Assay |
| Description: | Experimental Methods:1× activation buffer: 5 mM DTT 0.01% (V/V) Triton X-100 (preparation for current use);1× assay buffer: 50 mM NaCl 5 mM DTT 0.01% (V/V) Triton X-100 (preparation for current use);1× activation buffer was used to dilute recombinant human cathepsin C/DPP1 enzyme and recombinant human cathepsin L (rhCathepsin L) enzyme to concentrations of 2 ng/μL and 0.4 ng/μL, respectively; equal volume of two working solutions of enzyme was taken, mixed well and incubated at 25° C. for 60 minutes;the compounds to be tested were 5-fold diluted with a multi-channel pipette to the 8th concentration, i.e., diluted from 1 mM to 12.8 nM. Then 1× experimental buffer was used to dilute each compound to be tested by gradient into a 4% DMSO working solution. The working solution was added to corresponding wells for 5 μL/well, and duplicate experiment was set. The mixture was centrifuged at 1000 rpm for 1 minute;5 μL/well of the enzyme mixture after incubation was taken and added to the white microwell plate. At this time, the amount of DPP1 enzyme in each well was 5 ng; 1× experimental buffer in 5 μL/well was added to the blank control well;Gly-Arg-AMC (hydrochloride) was diluted to 25 μM with 1× assay buffer. The diluted mixture in 10 μL/well was added to a white microwell plate. The substrate concentration was 12.5 μM at this time, and the microplate was centrifuged in a centrifuge at 1000 rpm for 1 minute. The concentration of the compound decreased from 10 μM to 0.128 nM. After centrifugation, the microplate was covered with membrane and incubated at 25° C. for 60 minutes;after incubation, fluorescence detection was performed using a multi-label analyzer with an excitation wavelength of 360 nm and an emission wavelength of 460 nm. |
| Affinity data for this assay | |
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