Legumain Protein, Mouse (HEK293, C-His)

Materials
Activation buffer: 0.1 M NaOAc, 0.1 M NaCl, pH 4.5
Assay buffer: 50 mM MES, 250 mM NaCl, pH 5.5
Legumain Protein, Mouse (HEK293, C-His) (HY-P70216A)
Substrate: Z-Ala-Ala-Asn-AMC (HY-136626), dissolved in DMSO to 10 mM
Standard: 7-Amino-4-methylcoumarin (AMC, HY-D0027)

Procedure
1. Preparation of Standard Curve: Dilute AMC with assay buffer to concentrations of 10000, 5000, 2500, 1250, 625, 312, 156, 78, 39, 19.5, and 0 pmol respectively. Add 100 μL of each dilution to the wells of a black microplate. Read in kinetic mode for 5 minutes using excitation and emission wavelengths of 380 nm and 460 nm (top read). Generate a standard curve with the measured RFU as the ordinate and the amount of standard substance as the abscissa, and derive the standard curve equation.
2. First, reconstitute the test protein with sterile water to 500 μg/mL, then dilute it to 50 μg/mL with activation buffer, and incubate at 37°C for 4 hours.
3. Dilute the protein solution to 2 ng/μL with assay buffer.
4. Dilute the substrate to 200 μM with assay buffer.
5. Initiate the reaction by adding 50 μL of 2 ng/μL Mouse Legumain and 50 μL of 200 μM substrate to each well. For blank wells, add 50 μL of assay buffer and 50 μL of 200 μM substrate.
6. Read in kinetic mode for 5 minutes using excitation and emission wavelengths of 380 nm and 460 nm.
7. Calculate the specific activity: