Sigma-Aldrich ChemiSCREEN EP3 Prostanoid Receptor Membrane Preparation，用于放射性配体结合测定和GTP γ S结合的人EP3 GPCR膜制备。

产品概述

编码EP 3剪接变体6的全长人PTGER 3 cDNA

Prostanoids bind to a family of 8 GPCRs to exert their biological effects (Narumiya and FitzGerald, 2001). The prostaglandin PGE₂ causes pain, vasodilation, immunosuppression of T cells, bone resorption and promotion of carcinogenesis. Four related GPCRs, EP₁, EP₂, EP₃ and EP₄, each bind to PGE₂, but the different G protein coupling status of each receptor leads to distinct biological effects. Further diversity is generated by alternative splicing; the human gene for EP₃ generates 9 alternatively spliced mRNAs encoding 8 isoforms of EP₃ (Kotani et al., 1997). These isoforms of EP₃ vary in sequence at their C-termini, and differ in their ability to couple to Gs, Gq or Gi (Kotani et al., 1995). EP3 is required for fever induced by pyrogens, a response long attributed to prostaglandins by the antipyretic action of aspirin and other COX inhibitors (Ushikubi et al., 1998). Chemicon′s EP₃ 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 antagonists of EP₃ interactions and its ligands. The membrane preparations exhibit a Kd of 1.56 nM for [3H]-PGE₂. With 1.5 nM [3H]-PGE₂, 5 µg/well and 10 µg/well EP₃ Membrane Prep typically yield greater than 10 and 20-fold signal-to-background ratios, respectively.

应用领域

放射性配体结合测定和GTP结合。

生化/生理作用

GPCR Class：A

蛋白质靶标：EP 3

目标子家族：前列腺素类

产品特点

孵育条件将膜与放射性配体和未标记的竞争剂混合（检测浓度见图1和图2），孵育1-2小时。过滤前，用0.33%聚乙烯亚胺包被GF/C 96孔滤板30分钟，然后用50 mM HEPES（pH 7.4）洗涤，将结合反应转移至滤板，并洗涤3次结合缓冲液：50 mM Tris，pH 7.4，10 mM MgCl 2，1 mM EDTA，过滤并在4 ℃下储存（Perkin Elmer#：NET-428）洗涤缓冲液：50 mM Tris，pH 7.4，过滤并在4 ℃下储存。

物理形态

一个包装包含足够用于至少200次测定（单位）的膜，其中单位是将产生大于10倍信号的膜的量：具有1.5 nM 3 H标记的PGE 2的背景包装缓冲液中的液体：50 mM Tris pH 7.4，10%甘油和1% BSA，无防腐剂。将膜蛋白在Iml包装缓冲液中调节至0.5mg/ml，快速冷冻，并储存在-80 ° C。

制备/使用说明

在-70 ° C下冷冻保存长达2年。不要冷冻和解冻。

分析说明

信号：使用不同量的EP 3膜制备液在竞争性结合试验中获得的背景和特异性结合值：10 µ g/孔5 µ g/孔信号：背景38 21特异性结合（cpm）6，386 4，053质量标准：1单位= 10 μ g Bmax：15.0 pmol/mg Kd：1.56 nM

技术规格