Recombinant Human Sphingosine Kinase 1/SPHK1 Protein, CF

• Assay Buffer: 50 mM HEPES, 2 mM Adenosine 5’-Triphosphate (ATP) (Sigma, Catalog # A7699),  0.1% Triton® X-100, 30 mM MgCl₂, pH 7.5
• Recombinant Human Sphingosine Kinase 1/SPHK1  (rhSPHK1) (Catalog # 5536-SK)
• Substrate Buffer: 4 mg/mL BSA in deionized water
• Fluorogenic Substrate: NBD-Sphingosine (NBD-Sph) (Avanti Polar Lipids, Catalog # 810205X), 523 µM stock in DMSO
• Aqueous Extraction Buffer: 1.0 M KPO₄, pH 8.5
• Organic Extraction Buffer: chloroform:methanol (2:1, v/v)
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rhSPHK1 to 0.2 µg/mL in Assay Buffer.
2. Dilute Substrate to 30 µM in Substrate Buffer.
3. Mix 50 µL of 30 µM Substrate with 50 µL of the diluted rhSPHK1 in a microcentrifuge tube in triplicate. Include a blank consisting of 50 µL of 30 µM Substrate with 50 µL of Assay Buffer in triplicate.
4. Incubate for 30 minutes at room temperature.
5. After incubation, add 100 µL of Aqueous Extraction Buffer. Mix briefly, and then add 500 µL of Organic Extraction Buffer. Vortex at high speed for 30 seconds.
6. Centrifuge tubes in a microcentrifuge at top speed for 2 minutes to separate the phases.
7. Remove 200 µL of aqueous (upper) phase from each tube and load into a well of a plate.
8. Read the plate in endpoint mode at excitation and emission wavelengths of 481 nm and 542 nm, respectively.
9. Calculate specific activity:

     *Adjusted for Substrate Blank
     **Derived using calibration standard NBD-Sphingosine-1-Phosphate (Avanti Polar Lipids, Catalog # 810207X).

• rhSPHK1: 0.010 µg
• Substrate: 15 µM