Cathepsin L1 Protein, Human (HEK293, His, solution)

Materials
Assay buffer: 50 mM MES, 5 mM DTT, 1 mM EDTA, 0.005% (w/v) Brij-35, pH 6.0
Test protein: Cathepsin L1 Protein, Human (HEK293, His, solution) (HY-P7754)
Substrate: Z-Leu-Arg-AMC (HY-142021)
Standard: 7-Amino-4-methylcoumarin (AMC, HY-D0027)

Procedure
1. Standard Curve: Dilute AMC to concentrations of 100, 50, 25, 12.5, 6.25, and 0 μM using assay buffer. Add 50 μL of each dilution to black microplate wells. Read for 5 minutes in kinetic mode at excitation and emission wavelengths of 380 nm and 460 nm respectively. Plot the standard curve with the measured RFU values on the x-axis and the molar quantity of the standard substance on the y-axis to derive the standard curve equation.
2. Dilute Human Cathepsin L1 to 40 μg/mL using the assay buffer. 3. Incubate the diluted Human Cathepsin L1 on ice for 15 minutes. 4. Dilute Human Cathepsin L1 to 0.02 μg/mL using assay buffer. 5. Dilute the substrate to 80 μM using assay buffer. 6. Add different concentrations of Human Cathepsin L1 to a black microplate, 50 μL per well, and initiate the reaction by adding 50 μL of 80 μM substrate. Add 50 μL assay buffer and 50 μL 80 μM substrate to blank wells. 7. Read in kinetic mode at excitation wavelength 380 nm and emission wavelength 460 nm for 5 minutes.
8. Calculate specific activity: