Real-time imaging of single-molecule fluorescence with a zero-mode waveguide for the analysis of protein-protein interaction
Real-time imaging of single-molecule fluorescence with a zero-mode waveguide for the analysis of protein-protein interaction
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DOI：
10.1021/ac800726g
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发表时间：
2008-08-01
影响因子：
7.4
通讯作者：
Ohdomari, Iwao
中科院分区：
文献类型：
--
作者：
Miyake, Takeo;Tanii, Takashi;Ohdomari, Iwao
利用零模波导实现了单分子荧光的实时成像。通过修改ZMW几何形状，信号背景比是用常规ZMW可获得的信号背景比的两倍。改善的信号-背景比使得可以在5 μ M的浓度下观察伴侣蛋白GroEL和辅伴侣蛋白GroES之间的单个结合-释放事件。两个速率常数，代表两个计时器动力学释放GroES GroEL与ZMW测量，和测量同意与全内反射荧光显微镜。这些结果表明，新的ZMW使得在细胞内浓度下真实的时间直接观察蛋白质-蛋白质相互作用成为可能。
Real-time imaging of single-molecule fluorescence with a zero-mode waveguide (ZMW) was achieved. With modification of the ZMW geometry, the signal-to-background ratio is twice that obtainable with a conventional ZMW. The improved signal-to-background ratio makes it possible to visualize individual binding-release events between chaperonin GroEL and cochaperonin GroES at a concentration of 5 mu M. Two rate constants representing two-timer kinetics in the release of GroES from GroEL were measured with the ZMW, and the measurements agreed well with those made with a total internal reflection fluorescence microscopy. These results indicate that the novel ZMW makes feasible the direct observation of protein-protein interaction at an intracellular concentration in real time.