Recombinant Mouse Sphingosine Kinase 1/SPHK1 Protein, CF

• Assay Buffer: 50 mM HEPES, 250 mM NaCl, 2 mM ATP, 0.2% (v/v) Triton® X-100, 30 mM MgCl₂, pH 7.5
• Substrate Buffer: 4 mg/mL BSA in deionized water
• Aqueous Extraction Buffer: 1.0 M Potassium Phosphate, pH 8.5
• Organic Extraction Buffer: chloroform:methanol (2:1, v/v)
• Recombinant Mouse Sphingosine Kinase 1/SPHK1 (rmSPHK1) (Catalog # 6086-SK)
• Fluorogenic Substrate: NBD-Sphingosine (NBD-Sph) (Avanti Polar Lipids, Catalog # 810205P), 0.25 mg/mL (523 µM) stock in DMSO
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute Substrate to 30 µM in Substrate Buffer.
2. Dilute rmSPHK1 to 0.1 µg/mL in Assay Buffer.
3. Mix 50 μL of 30 µM Substrate with 50 µL of the diluted rmSPHK1 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 30 minutes at room temperature.
5. After incubation, add 100 µL of Aqueous Extraction Buffer to each reaction. Mix briefly and then add 500 µL of the Organic Extraction Buffer to each reaction.
6. Vortex at high speed for 30 seconds.
7. Centrifuge tubes in a microcentrifuge at top speed for 2 minutes to separate the phases.
8. Remove 200 µL of the aqueous (upper) phase from each tube and place into the well of a black microplate.
9. Read the plate in endpoint mode at excitation and emission wavelengths of 481 nm and 542 nm, respectively.
10. Calculate specific activity:

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

• rmSPHK1: 0.005 μg
• Substrate: 15 µM