HTRF术语详解 详细 >>
FRET：指荧光共振能量转移，在供体和受体相互靠得很近时，光子能从一个受激发的荧光团（供体）转移到另一个荧光团（受体），并使后者发出荧光。
HTRF：均相时间分辨荧光，Homogeneous Time-Resolved Fluorescence ）是用来检测纯液相体系中待测物的一种常用方法。
背景（Background）：指在没有加入compound时检测到的信号值。
荧光受体（acceptor）：指在荧光团受到激光激发时，能发出特定波长的发射光的基团。HTRF®的能量受体均可为XL665和d2。
资料下载 更多 >>
- New HTRF cellular platform for cell surface receptors' study and screening
- Homogeneous Time-Resolved Fluorescence Part1:Methodological aspects
- HTRF GPCR Solutions:Gi,Gs,Gq receptor screening using a single technology
- HTRF Compatible Readers:Unleash your lab's capacity to read HTRF technology
- Forget Elisa use HTRF biomarkers
Development of a Cyclic Adenosine Monophosphate Assay for G(i)-Coupled G Protein-Coupled Receptors by Utilizing the Endogenous Calcitonin Activity in Chinese Hamster Ovary Cells
By: Wang Y, Kong Y, Shei GJ, Kang L, Cvijic ME.
1 Metabolic Disease Research, Bristol-Myers Squibb Company , Pennington, New Jersey.
Abstract Activation of G(i)-coupled G protein-coupled receptor (GPCRs) by their ligands leads to inhibition of adenylyl cyclase (AC) and reduction of cyclic adenosine monophosphate (cAMP) levels in cells. The traditional cAMP assay for G(i)-coupled GPCRs commonly uses forskolin, a nonspecific AC activator, to increase the basal cAMP level in cells to create an assay window for ligand detection. However, there is still a need to develop a nonforskolin-based cAMP assay because of the challenges inherent in titrating the concentration of forskolin to achieve a reliable assay window, along with issues related to the cAMP-independent effects of forskolin. Herein, we describe such an assay by utilizing the endogenous activity of the calcitonin receptor in Chinese hamster ovary (CHO) cells. The calcitonin receptor is a G(s)-coupled GPCR that, when activated by calcitonin, leads to the stimulation of AC and increases cAMP in cells. Thus, we use calcitonin, instead of forskolin, to increase the basal cAMP level in CHO cells to achieve an assay window. We demonstrated that calcitonin peptides robustly increased cAMP accumulation in several CHO cell lines stably expressing well-known G(i)-coupled GPCRs, such as the Dopamine D2 receptor, the Opioid μ receptor, or the Cannabinoid receptor-1. Agonists of these G(i)-coupled GPCRs attenuated calcitonin-induced cAMP production in their receptor stable cell lines. On the other hand, antagonists and/or inverse agonists blocked the effects of their agonists on calcitonin-induced cAMP production. This calcitonin-based cAMP assay has been demonstrated to be sensitive and robust and exhibited acceptable assay windows (signal/noise ratio) and, thus, can be applied to screen for agonists and antagonists/inverse agonists of G(i)-coupled GPCRs in high-throughput screening formats.
编辑: cq
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