A highly sensitive protocol for microscopy of alkyne lipids and fluorescently tagged or immunostained proteins[S]
A highly sensitive protocol for microscopy of alkyne lipids and fluorescently tagged or immunostained proteins[S]
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炔烃脂质和荧光标记或免疫染色蛋白质显微镜检查的高灵敏度方案[S]
作者：
Anne Gaebler;Anke Penno;Lars Kuerschner;C. Thiele
研究特定脂质的细胞定位的需求导致了脂质探针和显微镜的最新进展。炔脂质带有一个小的，非干扰性的标签，可以检测到点击反应与叠氮化物偶联的报告。荧光炔脂质成像关键取决于适当的叠氮化物报告和标记协议，允许有效的点击反应，因此灵敏的检测。我们合成了几个叠氮化物报告与不同的间隔区组件，并测试其适用于固定细胞中的炔脂质成像。将铜螯合吡啶甲基部分实施到荧光或基于生物素的叠氮化物试剂中强烈增加了成像常规的灵敏度。我们证明了这种方法的适用性，并使用不同的脂质类和实验设置的性能进行评估。由于叠氮化物吡啶甲基报告物允许降低的铜催化剂浓度，因此它们还能够使炔脂质与包括增强的绿色荧光蛋白在内的多种荧光蛋白共成像。或者，正如我们也表明，显微镜下的炔脂质可以结合蛋白质检测免疫细胞化学。总之，我们提出了一个强大的，灵敏的，和高度通用的协议标记的炔脂类与叠氮化物偶联的报告荧光显微镜，可以结合不同的蛋白质检测和成像技术。
The demand to study the cellular localization of specific lipids has led to recent advances in lipid probes and microscopy. Alkyne lipids bear a small, noninterfering tag and can be detected upon click reaction with an azide-coupled reporter. Fluorescent alkyne lipid imaging crucially depends on appropriate azide reporters and labeling protocols that allow for an efficient click reaction and therefore a sensitive detection. We synthesized several azide reporters with different spacer components and tested their suitability for alkyne lipid imaging in fixed cells. The implementation of a copper-chelating picolyl moiety into fluorescent or biotin-based azide reagents strongly increased the sensitivity of the imaging routine. We demonstrate the applicability and evaluate the performance of this approach using different lipid classes and experimental setups. As azide picolyl reporters allow for reduced copper catalyst concentrations, they also enable coimaging of alkyne lipids with multiple fluorescent proteins including enhanced green fluorescent protein. Alternatively, and as we also show, microscopy of alkyne lipids can be combined with protein detection by immunocytochemistry. In summary, we present a robust, sensitive, and highly versatile protocol for the labeling of alkyne lipids with azide-coupled reporters for fluorescence microscopy that can be combined with different protein detection and imaging techniques.
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影响因子：
3.3
作者：
Klauda, Jeffery B.;Venable, Richard M.;Freites, J. Alfredo;O'Connor, Joseph W.;Tobias, Douglas J.;Mondragon-Ramirez, Carlos;Vorobyov, Igor;MacKerell, Alexander D., Jr.;Pastor, Richard W.
通讯作者：
Pastor, Richard W.
影响因子：
3.4
作者：
Kaiser, RD;London, E
通讯作者：
London, E
稳定同位素辅助脂质组学与非靶向同位素过滤相结合，是揭示脂质代谢复杂动力学的工具
影响因子：
7.4
作者：
Li, Jia;Hoene, Miriam;Xu, Guowang
通讯作者：
Xu, Guowang
影响因子：
16.6
作者：
Uttamapinant, Chayasith;Tangpeerachaikul, Anupong;Grecian, Scott;Clarke, Scott;Singh, Upinder;Slade, Peter;Gee, Kyle R.;Ting, Alice Y.
通讯作者：
Ting, Alice Y.
使用点击化学模块化合成生物活性磷脂酸探针。
影响因子：
--
作者：
Smith,MatthewD;Sudhahar,ChristopherG;Gong,Denghuang;Stahelin,RobertV;Best,MichaelD
通讯作者：
Best,MichaelD