ASAH2 Protein, Mouse (HEK293, His)

Materials
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)
ASAH2 Protein, Mouse (HEK293, His) (HY-P76735)
Substrate: C12-ceramide (Avanti Polar Lipids, 50 mM stock in Chloroform)
Standard: Sphingosine
o-Phthaldialdehyde (o-PA): 50 mg/mL stock in DMSO
F16 Black Maxisorp Plate

Procedure
1. Standard Curve: Dilute Sphingosine (standard) in sssay buffer to concentrations of 0, 1.5625, 3.125, 6.25, 12.5, 25, 50 μmol/L. Add 200 μL of each concentration to the wells of a black microplate. Read the fluorescence with excitation and emission wavelengths of 330 nm and 450 nm (top read). Plot the measured RFU on the y-axis and the concentration of the standard (μmol/L) on the x-axis to create the standard curve and obtain the standard curve equation.
2. Dissolve 10 μL of 50 mM substrate in 1.99 mL of assay buffer to achieve a concentration of 250 μM.
3. Dilute Mouse ASAH2 to 0.05 μg/mL in assay buffer.
4. Mix 200 μL of 250 μM substrate with 50 μL of 0.05 μg/mL Mouse ASAH2. Include one blank with 50 μL of Mouse ASAH2 and 200 μL of assay buffer, and another blank with 200 μL of substrate and 50 μL of assay buffer.
5. Incubate at 37°C for 1 hour, then heat at 95-100°C for 5 minutes to stop the reaction.
6. Dilute o-PA to 2 mg/mL in o-PA buffer.
7. Add 250 μL of o-PA mixture to all reactions and mix well.
8. Incubate at room temperature for 10 minutes.
9. Add 200 μL (in duplicate) of the reaction mixture and controls to the plate.
10. Read the fluorescence in endpoint mode at excitation and emission wavelengths of 330 nm and 450 nm, respectively. 11. Calculate specific activity: