Description
General description
Full-length human GPR43 cDNA
GPR43 is a GPCR that, along with GPR41, is activated by short chain carboxylic acids formate, acetate, proprionate, butyrate and pentanoate. Neutrophils and other leukocytes selectively express GPR43, and GPR43 mediates calcium flux and chemotaxis in these cells by coupling to both Gq and Gi (Brown et al., 2003; Le Poul et al., 2003). Millipore′s GPR43 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 GPR43 interactions with its ligands. The cell line exhibits a calcium response with EC50s of 11.8μM for sodium propionate. The membrane preparations exhibit EC50s of 40.7 μM for sodium propionate in a GTPγS binding assay.
Application
Radioligand binding assay and GTPγS binding
Biochem/physiol Actions
GPCR Class: A
Protein Target: FFA2 / GPR43
Target Sub-Family: Free Fatty Acid
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 MgCl2/0.5 μM GDP in a nonbinding 96-well plate. Unlabeled sodium propionate 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.
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 GPR43 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: Membranes protein were adjusted to 1 mg/mL in packaging buffer, and dispensed at 1 mL/vial. Vials were 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 sodium propionate: ~ 40.7 μM
Legal Information
CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany
GenBank is a registered trademark of United States Department of Health and Human Services
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.
