Recombinant Human Alkaline Phosphatase/ALPL Protein, CF

• 6 months from date of receipt, -20 to -70 °C as supplied.
• 3 months, -20 to -70 °C under sterile conditions after opening.

• Assay Buffer: 50 mM Tris, 1 mM MgCl₂, pH 9.0
• Recombinant Human Alkaline Phosphatase/ALPL/TNAP (rhTNAP) (Catalog # 2909-AP)
• Substrate: 4-Methumbelliferly phosphate (4-MUP) (Calbiochem, Catalog # 474431), 10 mM in deionized water
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rhTNAP to 0.01 ng/µL in Assay Buffer.
2. Dilute Substrate 50 µM in Assay Buffer.
3. In a plate load 50 µL of 0.01 ng/µL rhTNAP to wells. Include a Substrate Blank of 50 µL of Assay Buffer.
4. Start the reaction by adding 50 µL of 50 µM Substrate to wells.
5. Read at excitation and emission wavelengths of 365 nm and 445 nm (top read), respectively in kinetic mode for 5 minutes.
6. Calculate specific activity:

     *Adjusted for Substrate Blank

     **Derived using calibration standard 4-Methumbelliferone (Sigma, Catalog # M1381).

• rhTNAP: 0.0005 µg
• Substrate: 25 µM