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公开(公告)号：EP2329272A2
公开(公告)日：2011-06-08
申请号：EP09740420.6
申请日：2009-07-30
发明人： ZWIER, Jurriaan , POOLE, Robert , ANSANAY, Hervé , FINK, Michel , TRINQUET, Eric
IPC分类号： G01N33/566 , G01N33/58
CPC分类号： G01N33/542 , G01N33/5035 , G01N33/58 , G01N2458/40 , Y10T436/13
摘要： A method for detecting the internalization of a transmembrane protein of interest expressed at the surface of a cell, comprising the following steps: a) labelling the protein of interest with a fluorescent metal complex, the lifetime of which is greater than 0.1 ms; b) adding to the reaction medium a composition capable of causing the internalization of the protein of interest; c) adding to the reaction medium a modulating agent selected from: a. a fluorescent or nonfluorescent FRET acceptor compound compatible with said fluorescent metal complex, the final concentration of which in the reaction medium is greater than 10
‑7 M; b. a reducing agent, the redox potential of which is less than +0.1 V and preferably between 0.25 and 0.75 V; c. an agent which binds specifically, by noncovalent bonding, with the fluorescent metal complex; d. a metal ion which competes with the rare earth so as to form a nonfluorescent metal complex; d) measuring the luminescence emitted by the reaction medium at the emission wavelength of the fluorescent metal complex and/or at the emission wavelength of the modulating compound when said compound is a fluorescent acceptor compound; e) comparing the signal measured in step d) with a reference signal measured on cells having been subjected only to steps a) and c). Use: Method for detecting membrane protein internalization.摘要翻译： 一种检测细胞表面表达的感兴趣的跨膜蛋白的内化的方法，包括以下步骤：a）用荧光金属配合物标记感兴趣的蛋白质，其寿命大于0.1ms; b）向反应介质中加入能够引起感兴趣蛋白质内化的组合物; c）向反应介质中加入选自以下的调节剂：a。 与所述荧光金属配合物相容的荧光或非荧光FRET受体化合物，其在反应介质中的最终浓度大于10 -7 M; 湾 还原剂，其氧化还原电位低于+ 0.1V，优选在0.25和0.75V之间; C。 通过非共价键特异性结合荧光金属配合物的试剂; d。 与稀土竞争以形成无荧光金属络合物的金属离子; d）当所述化合物是荧光受体化合物时，在荧光金属络合物的发射波长处和/或在调制化合物的发射波长处测量由反应介质发射的发光; e）将步骤d）中测得的信号与仅在经过步骤a）和c）的细胞上测量的参考信号进行比较。 用途：检测膜蛋白内在化的方法。
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