Kallikrein-2 Protein, Human (HEK293, His)

Materials
Assay buffer: 50 mM Tris-HCl, 0.15 M NaCl, 10 mM CaCl?, 0.05% (w/v) Brij-35, pH 7.5
Test protein: Kallikrein-2 Protein, Human (HEK293, His)
Substrate: Z-Phe-Arg-AMC, 10 mM stock in DMSO
Standard: AMC, 10 mM stock in DMSO

Procedure
1. Prepare calibration curve: Dilute the standard solution to 3 μM with assay buffer, then perform six serial dilutions. Dispense 100 μL of each dilution into black microplate wells. Read in kinetic mode for 5 minutes at excitation (380 nm) and emission (460 nm) wavelengths (top read). Plot the standard curve with measured RFU on the y-axis and standard substance amount on the x-axis to obtain the standard curve equation.
2. Dilute Human Kallikrein-2 to 5 μg/mL in assay buffer.
3. Dilute substrate Z-Phe-Arg-AMC to 200 μM in assay buffer.
4. Instrument Settings: Select Kinetic and Fluorescence functions. For Kinetic mode, set 11 cycles with 30-second intervals between cycles. For Fluorescence, set excitation at 380 nm and emission at 460 nm. 5. Experimental group: 50 μL, 5 μg/mL protein + 50 μL, 200 μM substrate.
Blank group: 50 μL assay buffer + 50 μL, 200 μM substrate.
6. Reading: Place the microplate into the microplate reader for reading.
7. Calculate specific activity: