Sphingolipids |
| Sphingosine-1-Phosphate |
Purity
>99% |
Product Name |
Product Number |
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Sphingosine-1-phosphate |
860492 |
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The "sphingosin" backbone of sphingolipids was so
named by J. L. W. Thudichum in 1884 for its enigmatic ("Sphinxlike")
properties. Although still an elusive class of lipids, research
on the involvement of sphingolipids in the signal transduction
pathways that mediate cell growth, differentiation, multiple
cell functions, and cell death has been rapidly expanding our
understanding of these compounds. In addition to the newly discovered
role of ceramide as an intracellular second messenger for tumor
necrosis factoralpha, IL1beta, and other cytokines, sphingosine,
sphingosine1phosphate, and other sphingolipid metabolites have
recently been demonstrated to modulate cellular calcium homeostasis
and cell proliferation. The sphingolipid metabolite sphingosine1phosphate
(SPP) has been implicated as a second messenger in cell proliferation
and survival. However, many of its biological effects are due
to binding to unidentified receptors on the cell surface. Recent
evidence suggests that branching pathways of sphingolipid metabolism
may mediate either apoptotic or mitogenic responses depending
on the cell type and the nature of the stimulus. Sphingolipid
metabolites, such as ceramide and sphingosine1phosphate (SPP),
are emerging as a new class of second messengers involved in
cellular proliferation, differentiation, and apoptosis.
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Analysis by Thin Layer Chromatography
Reagents and Materials:
TLC Plate: Whatman K6 (normal phase silica)
Mobile Phase: Chloroform:Methanol:33% Dimethylamine (5:15:3, v/v)
Detection Method: Iodine vapors, Molybdate Spray (see http://www.avantilipids.com/TechnicalDetectionMethodsForThinLayerChromatography.html
for
Detection of Phosphorus)
Rf: ~0.35
Procedure:
Dissolve aliquot of S-1-P in mobile phase (should produce
a clear solution; see Note below).
Apply sample to TLC plate.
Develop plate using mobile phase listed above.
Use detection methods listed above to visualize sample.
Note:
Do not dissolve entire stock of S-1-P with mobile
phase solvent as this solvent
is not suitable for use with
cells.
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| References |
- Merrill AH Jr; Schmelz EM; Dillehay DL; Spiegel S; Shayman
JA; Schroeder J.J.; Riley R.T.; Voss K.A.; Wang E.; (1997)
Sphingolipidsthe enigmatic lipid class: biochemistry, physiology,
and pathophysiology. Toxicol Appl Pharmacol, 142:1,
20825
- Lee MJ; Van Brocklyn J.R.; Thangada S.; Liu C.H.; Hand
A.R.; Menzeleev R.; Spiegel S.; Hla T., (1998) Sphingosine-1phosphate
as a ligand for the G proteincoupled receptor EDG1. Science, 279:5356, 15525
- Spiegel S; Cuvillier O.; Edsall L.C.; Kohama T.; Menzeleev
R.; Olah Z.; Olivera A.; Pirianov G.; Thomas D.M.; Tu Z.;
Van Brocklyn J.R.; Wang F.,(1998) Sphingosin1phos-phate
in cell growth and cell death. Ann N. Y. Acad. Sci., 845:, 118Edsall L.C.; Pirianov G.G.; Spiegel S., (1997)
Involvement of sphingosine-1phosphate in nerve growth factormediated
neuronal survival and differentiation. J Neurosci, 17:18,
695260
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Sample Preparation For Mass Spectometry
|
Dissolve approximately 1 mg sphingosine-1-phosphate in
1 mL 65:35:8:0.1 chloroform: methanol: water: (ammonium hydroxide
+ 10 mM ammonium acetate).
It may be necessary to vortex and/or sonicate
the sample until it is completely in solution.
The above solution is then further diluted in a solution
of 70:30:0.1 acetonitrile: water: (ammonium hydroxide
+ 10 mM ammonium acetate).
The final concentration was 2.6 pmol/uL (1 ug/mL). |
Electrospray negative ionization mass spectrum
of Sphingosine-1-Phosphate.
enlarge
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Sphingosine-1-Phosphate
Solubility Protocol & Cell Delivery
Organic Solutions:
The solubility of stock solutions of sphingoid base 1-phosphates
may be increased by the addition of dimethylamine.
Water Solutions:
Solubility of S1P is difficult using organic solvents
so typically it is suspended in methanol to aliquot, the methanol
dried and the residue dispersed using fatty acid free BSA
solution.
A procedure for preparation of a S1P stock solution follows:
Stock Solution:
125µM Sphingosine-1-Phosphate in essentially fatty
acid free Bovine Serum Albumin (BSA, 4mg/ml)
Preparation of Stock Solution:
Methanol Solution:
- Add Sphingosine-1-phosphate to methanol:water 95:5 (0.5mg/ml)
- Heat mixture (45°C-65°C methanol boiling point)
and sonicate until Sphingosine-1-Phosphate is suspended
(solutions are not clear - forms a slightly hazy suspention).
- Transfer desired amounts of methanol:water stock solution
to glass vessel.
- Remove methanol:water solution using a stream of dry nitrogen.
- Place glass vessel into a warm (approximately 50°C)
water bath.
- Rotate glass vessel during evaporation to make thin film
of Sphingosine-1-phosphate on tube wall.
- Store Sphingosine-1-Phosphate at -20°C in closed container.
Maximum storage one year.
BSA Solution
- Add enough BSA (4mg/ml) in water at 37°C to glass
vessel containing Sphingosine-1-Phosphate
to make a 125µM Sphingosine-1-Phosphate solution.
Incubate for 30 minutes and vortex occasionally.
Maximum storage three months.
Reference List
BOOKS
PAPERS
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