| Assay Method Information | |
| | CTSC Inhibition Assay (In Vitro) |
| Description: | The test specimen was dissolved in dimethylsulfoxide (DMSO) at 10 mmol/L to prepare a stock solution. In a case where a solution in which the stock solution was diluted 15-fold with DMSO was used as the maximum evaluation concentration (0.667 mmol/L), 5-fold dilution was repeated in DMSO from the solution to prepare a serially diluted solution. 248.8 μL of the CTSC assay buffer was added to 1.12 μL of the serially diluted sample prepared in the DMSO to prepare a solution of a CTSC inhibition assay compound.The final composition of the CTSC inhibition assay mixture was 0.66 ng/mL activated CTSC, 50 mmol/L MES/NaOH (pH: 5.0), 50 mmol/L NaCl, 0.001% Triton X-100, 5 mmol/L DTT, 30 μmol/L GF-AFC, and 0.15% DMSO, and the procedure was as follows. That is, 4 μL of the compound solution and 4 μL of the 2 ng/mL activated CTSC solution were mixed with each other in a 384 well plate and incubated at 25° C. for 30 minutes. Thereafter, 4 μL of the GF-AFC solution dissolved in the CTSC assay buffer was added to the mixture so as to be 90 μmol/L. After mixing to be uniform, a fluorescence emission intensity at 505 nm in a case of being excited at 390 nm after incubating at 25° C. for 1 hour was measured with a plate reader. A 4-parameter regression analysis was performed with GraphPad Prism (manufactured by GraphPad Software) from a plot of the fluorescence intensity at 505 nm and the sample concentration, and an IC50 value was calculated. |
| Affinity data for this assay | |
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