Recombinant Mouse Sphingosine Kinase 2/SPHK2 Protein, CF Summary
Ala2-Ala617, with an N-terminal Met and an N-terminal 7-His tag
Accession # Q9JIA7
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
|Formulation||Supplied as a 0.2 μm filtered solution in HEPES, NaCl, DTT, Glycerol and Tween® 20.|
|Shipping||The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.|
|Stability & Storage:||Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
- Assay Buffer: 50 mM HEPES, 1.0 M NaCl, 2 mM DTT, 2 mM ATP, 0.1% Triton® X-100, 20 mM MgCl2, pH 7.5
- Substrate Buffer: 4 mg/mL BSA in deionized water
- Aqueous Extraction Buffer: 1.0 M KH2PO4, pH 8.5
- Organic Extraction Buffer: chloroform:methanol (2:1, v/v)
- Recombinant Mouse Sphingosine Kinase 2/SPHK2 (rmSPHK2) (Catalog # 6058-SK)
- Fluorogenic Substrate: NBD-Sphingosine (NBD-Sph) (Avanti Polar Lipids, Catalog # 810205P), 0.5 mg/mL (0.523 µM) stock in DMSO
- Adenosine 5′-triphosphate (ATP) (Sigma, Catalog # A-7699), 10 mM in deionized water
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute Substrate to 30 µM in Substrate Buffer.
- Dilute rmSPHK2 to 0.4 µg/mL in Assay Buffer.
- Mix 50 μL of 30 µM Substrate with 50 µL of the diluted rmSPHK2 in a microcentrifuge tube in duplicate.
- Include a blank consisting of 50 µL of 30 µM Substrate with 50 µL of Assay Buffer in duplicate.
- Incubate 30 minutes at room temperature.
- 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. Vortex at high speed for 30 seconds.
- Centrifuge tubes in a microcentrifuge at top speed for 2 minutes to separate the phases.
- Remove 200 µL of the aqueous (upper) phase from each tube and place into the well of a microplate.
- Read the plate in endpoint mode at excitation and emission wavelengths of 481 nm and 542 nm, respectively.
- Calculate the specific activity:
Specific Activity (pmol/min/µg) =
|Adjusted Fluorescence* (RFU) x Conversion Factor** (pmol/RFU)|
|Incubation time (min) x amount of enzyme (µg)|
*Adjusted for Substrate Blank
**Derived using calibration standard NBD-Sphingosine-1-Phosphate (Avanti Polar Lipids, Catalog # 810207X).
- rmSPHK2: 0.020 μg
- Substrate: 15 µM
Background: Sphingosine Kinase 2/SPHK2
Sphingosine kinases catalyze the phosphorylation of sphingosine to sphingosine-1-phosphate, a lipid messenger molecule involved in the regulation of processes such as growth, differentiation, and cellular migration (1). Two types of sphingosine kinases, SPHK1 and SPHK2, are known to be expressed in human cells. The two enzymes share considerable amino acid sequence similarity, but differ in their N-terminal and central regions (2). The two proteins also differ in tissue distribution and some kinetic properties (2). Sphingosine kinases can be either cytosolic or membrane-associated. Differences in the distribution of SPHK1 and SPHK2 within cells may be the cause of differences that have been observed in the function of the two enzymes, particularly their roles in apoptosis (3). Sphingosine kinases are considered to be therapeutic targets for cancer and immune system disorders (4, 5).
- Spiegel, S. (1999) J. Leukocyte Biol. 65:341.
- Liu, H. et al. (2000) J. Biol. Chem. 275:19513.
- Maceyka, M. et al. (2005) J. Biol. Chem. 280:37118.
- Shida, D. et al. (2008) Curr. Drug Targets 9:622.
- Melendez, A.J. (2008) Biochim. Biophys. Acta 1784:66.
Product Specific NoticesCoomassie is a registered trademark of Imperial Chemical Industries Ltd. Tween is a registered trademark of ICI Americas. Triton is a registered trademark of Union Carbide Corp.
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