PEPD Protein, Human

Materials
Activation buffer: 0.05 M Tris, 0.01 M CaCl₂, 0.15 M NaCl, 0.05% Brij-35, MnCl₂, pH 7.5
Assay buffer: 0.05 M Tris, 0.1 M NaCl, 0.01% Brij-35, pH 8.5
PEPD Protein, Human (HY-P70992)
Substrate: Gly-Pro-AMC (HY-137834)
Standard: 7-amino, 4-Methyl Coumarin

Procedure
1. Dilute standard in assay buffer to concentrations of 0, 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5, 5, 10, 20, 40, 80 μM.
2. Add 100 μL of each standard concentration to the wells.
3. Read the fluorescence in kinetic mode for 5 minutes with excitation and emission wavelengths of 380 nm and 460 nm (top read) .
4. Plot the standard concentrations on the y-axis and the measured RFU on the x-axis to create the standard curve and obtain the standard curve equation.
5. Dilute Human PEPD to 0.2 μg/mL in activation buffer.
6. Dilute the substrate to 50 μM in assay buffer.
7. Add 50 μL of 0.2 μg/mL Human PEPD to the wells, followed by 50 μL of 50 μM substrate to initiate the reaction.
For the control group, add 50 μL of assay buffer and 50 μL of 50 μM substrate.
8. Read the fluorescence in kinetic mode for 5 minutes with excitation and emission wavelengths of 380 nm and 460 nm (top read).
9. Calculate specific activity: