Recombinant Human SMPD1 Protein, CF

• 6 months from date of receipt, -70 °C as supplied.
• 3 months, -70 °C under sterile conditions after opening.

• Assay Buffer: 50 mM MES, 0.5 µM ZnCl₂, pH 6.5
• Developing  Buffer: 0.2 M NaOH
• Recombinant Human SMPD1 (rhSMPD1) (Catalog # 5348-PD)
• Substrate: 2-N-Hexadecanoyl-4-nitrophenylphosphorylcholine (HNPPC) (Gojira Fine Chemicals, Catalog # HN1004), 50 mM stock solution in methanol.  Note: Incubate at 37 °C for 5 minutes and vortex to dissolve.
• 96-well Clear Plate (Catalog # DY990)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute rhSMPD1 to 2 µg/mL in Assay Buffer and incubate at 37 °C for 10 minutes.
2. Dilute Substrate to 1 mM in Assay Buffer and incubate at 37 °C for 10 minutes.
3. Combine 50 µL of rhSMPD1 and 50 µL of Substrate in a plate. Create Substrate Blanks by using Assay Buffer in place of rhSMPD1.
4. Cover plate and incubate at room temperature for 20 minutes in the dark.
5. Stop the reaction by adding 100 µL of Developing Buffer to each well.
6. Read (top read) absorbance in endpoint mode at 410 nm.
7. Calculate specific activity:

     *Adjusted for Substrate Blank

     **Derived using calibration standard p-Nitrophenol (Sigma-Aldrich, Catalog # 241326).

• rhSMPD1: 0.10 µg
• Substrate: 250 µM