Recombinant Human Sphingosine Kinase 1/SPHK1 Protein, CF Summary
Asp2-Leu398 with an N-terminal Met and 6-His tag
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 MES, NaCl, Glycerol, DTT and Triton®.|
|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, 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
- Dilute rhSPHK1 to 0.2 µg/mL in Assay Buffer.
- Dilute Substrate to 30 µM in Substrate Buffer.
- 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.
- Incubate for 30 minutes at room temperature.
- 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.
- Centrifuge tubes in a microcentrifuge at top speed for 2 minutes to separate the phases.
- Remove 200 µL of aqueous (upper) phase from each tube and load into a well of a plate.
- 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).
- rhSPHK1: 0.010 µg
- Substrate: 15 µM
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.
Citation for Recombinant Human Sphingosine Kinase 1/SPHK1 Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
1 Citation: Showing 1 - 1
A selective inhibitor of ceramide synthase 1 reveals a novel role in fat metabolism
Authors: N Turner, XY Lim, HD Toop, B Osborne, AE Brandon, EN Taylor, CE Fiveash, H Govindaraj, JD Teo, HP McEwen, TA Couttas, SM Butler, A Das, GM Kowalski, CR Bruce, KL Hoehn, T Fath, C Schmitz-Pe, GJ Cooney, MK Montgomery, JC Morris, AS Don
Nat Commun, 2018;9(1):3165.
Sample Types: Recombinant Protein
Applications: Enzyme Assay
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