Recombinant Mouse ASAH2 Protein, CF

• Assay Buffer: 25 mM MES, 150 mM NaCl, 1% (w/v) Sodium Cholate, pH 6.5
• o-PA Buffer: 0.2 M NaOH, 0.1% beta -mercaptoethanol (v/v)
• Recombinant Mouse ASAH2/N‑acylsphingosine Amidohydrolase-2 (rmASAH2) (Catalog # 3558-AH)
• Substrate: C12-ceramide (Avanti Polar Lipids, Catalog # 860512P), 50 mM stock in Chloroform
• 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. Dissolve 10 µL of 50 mM stock of Substrate in 1.99 mL Assay Buffer for a 250 µM concentration. (Note: Preheat assay buffer to
   37 °C and vortex for 30 seconds to dissolve Substrate).
2. Dilute rmASAH2 to 0.05 µg/mL in Assay Buffer.
3. Combine 200 µL of 250 µM Substrate and 50 µL of 0.05 µg/mL rmASAH2. Include one blank containing 50 µL rmASAH2 and 200 µL Assay Buffer and another blank containing 200 µL Substrate and 50 µL Assay Buffer.
4. Incubate at 37 °C for 1 hour.
5. Stop reactions by heating them at 95-100 °C for 5 minutes.
6. Dilute o-PA to 2 mg/mL in o-PA Buffer.
7. Add 250 µL of the o-PA mixture to all reaction vials, including controls. Mix well.
8. Incubate at room temperature for 10 minutes. Note: It is important not to deviate from this incubation time.
9. Load 200 µL (in duplicate) of reaction mixtures and controls in a plate.
10. Read at excitation and emission wavelengths of 330 nm and 450 nm (top read), respectively, in endpoint mode.
11. Calculate specific activity using the following equation:

     *Average duplicates, use the control with the higher RFU value to adjust fluorescence.
     **Derived using calibration standard Sphingosine (Avanti Polar Lipids, Catalog # 860490P).

• rmASAH2: 0.001 µg
• C12-ceramide: 100 μM
• o-PA: 1 mg/mL