Recombinant Mouse ASAHL Protein, CF

• Assay Buffer: 0.1 M Sodium Acetate, 0.1% (v/v) NP-40 substitute (Fluka, Catalog # 74385), pH 4.0
• Recombinant Mouse ASAHL/N‑acylethanolamine-hydrolyzing Acid A (rmASAHL) (Catalog # 4886-AH)
• Palmitoyl Ethanolamide (PEA) (Tocris, Catalog # 0879), 25 mM stock in dimethyl formamide
• Dithiothreitol (DTT) (Sigma, Catalog # D0632), 1 M stock in deionized water
• Sodium Hydroxide
• beta -mercaptoethanol (Sigma, Catalog # M7154)
• 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. Dissolve 10 µL of 25 mM stock into 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 rmASAHL to 1.25 µg/mL in Assay Buffer.
3. Set up reactions in 1.5 mL microtubes. Mix 200 µL of 50 µM PEA, 50 µL of 1.25 µg/mL rmASAHL, and 2.5 µL of 1 M DTT.
4. Incubate reaction tubes at 37 °C for 1 hour.
5. Prepare an o-PA solution. Mix 3.84 mL of 0.2 M Sodium Hydroxide, 4 µL beta -mercaptoethanol, and 160 µL 50 mg/mL o-PA.
6. Add 250 µL of the o-PA mixture (step 5) to all reaction vials. Mix well and incubate at room temperature for 10 minutes.
7. Create a control vial by combining, in this order, 250 µL o-PA mixture (step 5), 50 µL of 1.25 µg/mL rmASAHL, and 200 µL of 50 µM PEA.
8. Load in a plate 200 µL in duplicate of reaction mixtures and control.
9. Read at excitation and emission wavelengths of 330 nm and 450 nm (top read), respectively, in endpoint mode.
10. Calculate specific activity (Average duplicates):

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

• rmASAHL: 0.025 µg
• PEA: 20 µM
• o-PA: 1 mg/mL