ChemiSCREEN Membrane Preparation Recombinant Human GPR41 Free Fatty Acid Receptor

Full-length human GPR41 cDNA

GPR41 is a GPCR that, along with GPR43, is activated by short chain carboxylic acids formate, acetate, proprionate, butyrate and pentanoate (Brown et al., 2003; Brown et al., 2005). Binding of these ligands to GPR41 selectively activates Gi to inhibit cAMP accumulation. Expression of GPR41 is prominent in adipose tissue, increases during differentiation of cultured adipocytes, and allows short chain carboxylic acids to stimulate leptin synthesis in adipocytes (Xiong et al., 2003). Millipore′s GPR41 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 GPR41 interactions with its ligands. The cell line exhibits a calcium response with EC50s of 51.5μM for sodium propionate. The membrane preparations exhibit EC50s of 30.1 μM for sodium propionate in a GTPγS binding assay.

Radioligand binding assay and GTPγS binding

1 unit = 5 μg
EC50 in GTPγS binding assay by sodium propionate: ~ 30.1 μM

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.

Maintain frozen at -70°C for up to 2 years. Do not freeze and thaw.

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 GPR41 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.

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.