Recombinant Mouse Sphingosine Kinase 1/SPHK1 Protein, CF
Recombinant Mouse Sphingosine Kinase 1/SPHK1 Protein, CF Summary
Product Specifications
Glu2-Pro382, with an N-terminal Met and 6-His tag
Analysis
Product Datasheets
Carrier Free
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.
6086-SK
| Formulation | Supplied as a 0.2 μm filtered solution in MES, NaCl, Glycerol and DTT. |
| 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 Procedure
- 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
- Dilute Substrate to 30 µM in Substrate Buffer.
- Dilute rmSPHK1 to 0.1 µg/mL in Assay Buffer.
- 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.
- 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 black microplate.
- Read the plate in endpoint mode at excitation and emission wavelengths of 481 nm and 542 nm, respectively.
- Calculate 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). Per Reaction:
- rmSPHK1: 0.005 μg
- Substrate: 15 µM
Reconstitution Calculator
Background: Sphingosine Kinase 1/SPHK1
Sphingosine kinases are cytosolic or membrane-associated enzymes that catalyze the phosphorylation of sphingosine to sphingosine-1-phosphate (S1P). 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 (1). The two proteins also differ in tissue distribution and some kinetic properties (1). S1P is a lipid messenger that regulates diverse physiological processes including cell proliferation, migration, apoptosis, inflammation, calcium homeostasis and cytoskeletal structure (2, 3). The level of S1P is tightly controlled by SPHKs and S1P degrading enzymes. SPHK1 and its activation can be stimulated by several growth factors such as tumor necrosis factor-alpha, epidermal growth factor and transforming growth factor-beta (3, 4). Expression of SPHK1 has been found to increase in many human solid tumors and overexpression of SPHK1 is associated with tumor angiogenesis (5). Such studies have implicated SPHK1 as a new target for cancer treatment.
- Liu, H. et al. (2000) J. Biol. Chem. 275:19513.
- Spiegel, S. (1999) J. Leukocyte Biol. 65:341.
- Alemany, R. et al. (2007) Naunyn-Schmiedegerg’s Arch. Pharmacol. 374:413.
- Pederson, L. et al. (2008) Proc. Natl. Acad. Scis USA. 105:20764.
- Shida, D. et al. (2008) Curr. Drug Targets. 9:662.
Product Specific Notices
Coomassie is a registered trademark of Imperial Chemical Industries Ltd. Triton is a registered trademark of Union Carbide Corp.FAQs
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