Description
General description
Full-length human EDG1 cDNA encoding S1P₁
Sphingosine 1-phosphate (S1P) is a biologically active lysophospholipid that transmits signals through a family of five G-protein-coupled receptors to regulate cell proliferation, migration, cytoskeletal organization, and differentiation. S1P₁ was the first identified S1P receptor. It primarily couples to PTX-sensitive Gi/o proteins and mediates S1P-induced adenylate cyclase inhibition. Expression of S1P₁ is pervasive, including spleen, brain, heart, lung, adipose tissues, liver, thymus, kidney, and skeletal muscle (Zhang et al. 1999). The deletion of S1P₁ in mice results in embryonic lethality (Liu et al., 2000) with death attributable to incomplete vascular maturation. Recent reports demonstrate specific roles for S1P₁ in lymphocyte recirculation/egress (Matloubian et al., 2004). Millipore′s S1P₁ membrane preparations are crude membrane preparations made from our proprietary stable recombinant cell lines to ensure high-level of GPCR surface expression; thus, they are ideal HTS tools for screening of S1P₁ interaction with its ligands. The cell line exhibits a calcium response with EC50s of 141 μM for Sphingosine 1-phosphate. The membrane preparations exhibit EC50s of 9.5 μM for Sphingosine 1-phosphate in a GTPγS binding assay.
Application
Human S1P1 / EDG1 GPCR membrane preparation for Radioligand binding Assays & GTPγS binding.
Radioligand binding assay and GTPγS binding
Biochem/physiol Actions
GPCR Class: A
Protein Target: S1P1 / EDG1
Target Sub-Family: Lysophospholipid
Features and Benefits
Inucbation Conditions
Membranes are permeabilized by addition of saponin to an equal concentration by mass, then mixed with [³⁵S]-GTPγS (final concentration of 0.3 nM) in 20 mM HEPES, pH 7.4/100 mM NaCl/10 mM MgCl₂/10 μM GDP in a nonbinding 96-well plate. Unlabeled Sphingosine 1-phosphate was added to the final concentration indicated in Figure 1 (final volume 100 μL), and incubated for 30 min at 30°C. The binding reaction is transferred to a GF/B filter plate (Millipore MAHF B1H) previously prewetted with water. The plate is washed 3 times (1 mL per well per wash) with cold 10 mM sodium phosphate, pH 7.4, then dried and counted.
Note: Performing the reaction with lower GDP concentrations (0.5 μM) results in elevated constitutive activity (Figure 2).
One vial contains enough membranes for at least 200 assays (units), where one unit is the amount of membrane that will yield greater than 1000 cpm specific sodium propionate-stimulated [³⁵S]-GTPγS binding.
The S1P1 membrane preparation is expected to be functional in a radioligand binding assay; however, the end user will need to determine the optimal radiolabeled ligand for use with this product.
Physical form
Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA with no preservatives.
Packaging method: Membrane protein was adjusted to 1 mg/ml in packaging buffer, rapidly frozen, and stored at -80°C.
Preparation Note
Maintain frozen at -70°C for up to 2 years. Do not freeze and thaw.
Analysis Note
1 unit = 5 μg
EC50 in GTPγS binding assay by Sphingosine 1-phosphate: ~ 9.5 nM
Legal Information
CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany
Disclaimer
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
