Alkaline Phosphatase/ALPI Protein, Human (HEK293, His)

Materials
Assay buffer: 50 mM Tris, 1 mM MgCl₂, pH 9.0
Alkaline Phosphatase/ALPI Protein, Human (HEK293, His) (HY-P72822)
Substrate: 4-Methylumbelliferyl phosphate, dissolved in deionized water to 10 mM
Standard: 4-Methumbelliferone

Procedure
1. Standard Curve: Dilute 4-Methylumbelliferone in assay buffer to concentrations of 0, 6.25, 12.5, 25, 50, 100 μmol/L. Add 100 μL of each concentration to the wells of a black microplate. Read the fluorescence with excitation and emission wavelengths of 365 nm and 445 nm (top read). Plot the measured RFU on the y-axis and the concentration of the standard (μmol/L) on the x-axis to create the standard curve and obtain the standard curve equation.
2. Dilute Human ALPI to 0.1 μg/mL in assay buffer.
3. Dilute the substrate to 50 μM in assay buffer.
4. Experimental group: Add 50 μL of 0.1 μg/mL ALPI to the wells and initiate the reaction by adding 50 μL of 50 μM substrate.
Control group: Add 50 μL of assay buffer and 50 μL of 50 μM substrate.
5. Read the fluorescence in kinetic mode for 5 minutes with excitation and emission wavelengths of 365 nm and 445 nm, respectively.
6. Calculate specific activity: