Recombinant Human PLA2G4A 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, 500 mM NaCl, 1 mg/mL BSA, pH 8.5
• Substrate Buffer: 50 mM Tris, 500 mM NaCl, 20 mM CaCl₂, 1 mg/mL BSA, pH 8.5
• Recombinant Human PLA2G4A (rhPLA2G4A) (Catalog # 6659-PL)
• Substrate: 1-Hexadecanoyl-2-(1-Pyrenedecanoyl)-sn-Glycero-3-Phosphocholine (Invitrogen, Catalog # H361), Dilute to 2 mM in DMSO, then dilute to a final stock concentration of 400 µM in ethanol
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Thaw Substrate at 37 °C for five minutes. Mix well.
2. Dilute rhPLA2G4A to 2 µg/mL in Assay Buffer.
3. Dilute Substrate to 10 µM in Substrate Buffer.
4. Load 50 µL of 2 µg/mL rhPLA2G4A into a black well plate, and start the reaction by adding 50 µL of 10 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 10 µM Substrate.
5. Read at excitation and emission wavelengths of 345 nm and 395 nm (top read), respectively, in kinetic mode for 5 minutes.
6. Calculate specific activity:

   *Adjusted for Substrate Blank
   **Derived using calibration standard 1-pyrenedecanoic acid (Invitrogen, Catalog # P31).

• rhPLA2G4A: 0.10 µg
• Substrate: 5 µM