Recombinant Human ASAHL Protein, CF

• Assay Buffer: 0.1 M NaOAc, 0.1% (v/v) NP-40 substitute (Fluka, Catalog # 74385), pH 4.0
• Recombinant Human ASAHL/N‑acylethanolamine-hydrolyzing Acid A (rhASAHL) (Catalog # 4494-AH)
• Palmitoyl Ethanolamide (PEA) (Tocris, (Tocris, Catalog # 0879), 25 mM stock in dimethyl formamide
• Dithiothreitol (DTT) (Sigma, Catalog # D0632), 1 M stock in deionized water
• NaOH (Sigma, Catalog # 221465), 2 M stock in deionized water
• beta -mercaptoethanol (Sigma, Catalog # M-7154)
• o-phthaldialdehyde (o-PA) (Sigma, Catalog # P0657), 50 mg/mL stock in DMSO
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute PEA to 50 µM in Assay Buffer by dissolving 10 µL of 25 mM stock in 4.99 mL of Assay Buffer (Note: Preheat assay buffer to 37 °C and vortex for 30 seconds to completely solubilize the PEA).
2. Dilute rhASAHL to 1.25 µg/mL in Assay Buffer.
3. Combine 200 µL of 50 µM PEA, 50 µL of 1.25 µg/mL rhASAHL, and 2.5 µL of 1 M DTT.
4. Incubate reaction tubes at 37 °C for 1 hour.
5. Dilute NaOH to 0.2 M in deionized water.
6. Combine 3.84 mL of 0.2 M NaOH with 4 µL beta -mercaptoethanol and 160 µL of 50 mg/mL o-PA.
7. Stop the reactions by adding 250 µL of the o-PA mixture (step 6) to all the vials and mix well.
8. Incubate at room temperature for 10 minutes.
9. Combine 250 µL of o-PA mixture, 50 µL of 1.25 µg/mL rhASAHL, and 200 µL of 50 µM PEA in this order for a control.
10. Load 200 µL of reaction mixtures and control in a plate.
11. Read at excitation and emission wavelengths of 330 nm and 450 nm (top read), respectively, in endpoint mode.
12. Calculate specific activity (Average duplicates):

     *Adjusted for Control
     **Derived using calibration standard ethanolamine (Sigma, Catalog # E9508).

Per Well:

• rhASAHL: 0.025 µg
• Palmitoyl Ethanolamide: 20 µM
• o-PA: 1 mg/mL