使用 ELISA 和 cAMP 测量法鉴定瞬时转染细胞中的 G 蛋白和 G 蛋白偶联受体的方案。
Protocol to characterize G and G protein-coupled receptors in transiently transfected cells using ELISA and cAMP measurements.
机构信息
Rudolf-Schönheimer Institute for Biochemistry, Faculty of Medicine, Leipzig University, Johannisallee 30, 04103 Leipzig, Germany.
Rudolf-Schönheimer Institute for Biochemistry, Faculty of Medicine, Leipzig University, Johannisallee 30, 04103 Leipzig, Germany.
出版信息
STAR Protoc. 2023 Mar 17;4(1):102120. doi: 10.1016/j.xpro.2023.102120. Epub 2023 Feb 14.
Activation of G or G protein-coupled receptors (GPCRs) leads to changes of intracellular cyclic adenosine monophosphate (cAMP) levels. This protocol describes steps for cloning HA- and FLAG-tagged GPCRs, transient transfection of CHO-K1 or HEK293-T cells, and determination of basal and ligand-induced changes in intracellular cAMP levels. We detail enzyme-linked immunosorbent assays to determine relative GPCR plasma membrane and total expression levels. For complete details on the use and execution of this protocol, please refer to Schulze et al. (2022)..
G 蛋白或 G 蛋白偶联受体 (GPCR) 的激活会导致细胞内环腺苷酸 (cAMP) 水平的变化。本方案描述了 HA 和 FLAG 标记的 GPCR 克隆、CHO-K1 或 HEK293-T 细胞的瞬时转染，以及测定细胞内 cAMP 水平的基础和配体诱导变化的步骤。我们详细介绍了酶联免疫吸附测定法，以确定相对 GPCR 质膜和总表达水平。有关本方案使用和执行的完整详细信息，请参阅 Schulze 等人。(2022)。
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