Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy
Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy
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DOI：
10.1006/meth.2001.1189
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发表时间：
2001-07-01
期刊：
影响因子：
4.8
通讯作者：
Kenworthy, AK
中科院分区：
文献类型：
--
作者：
Kenworthy, AK
荧光共振能量转移（FRFT）检测荧光标记分子的接近距离超过100埃。当在荧光显微镜下进行时，FRET可以用来绘制体内蛋白质之间的相互作用。我们在这里描述了一种FRET显微镜方法，可用于确定在光显微镜水平上共定位的蛋白质是否相互作用。这种方法可以使用数字显微镜系统，如共聚焦显微镜或宽视场荧光显微镜耦合到电荷耦合器件（CCD）相机来实现。它很容易应用于用标准免疫荧光技术制备的样品，使用荧光染料标记的抗体作为FRET的供体和受体对。通过比较受体完全光漂白前后的供体荧光强度，基于FRET引起的供体荧光猝灭释放来量化能量转移效率。如前所述，该方法使用Cy3和Cy5作为供体和受体荧光团，但可以适用于其他FRET对，包括青色荧光蛋白和黄色荧光蛋白，(C) 2001学术出版社。
Fluorescence resonance energy transfer (FRFT) detects the proximity of fluorescently labeled molecules over distances > 100 Angstrom. When performed in a fluorescence microscope, FRET can be used to map protein-protein interactions in vivo. We here describe a FRET microscopy method that can be used to determine whether proteins that are colocalized at the level of light microscopy interact with one another. This method can be implemented using digital microscopy systems such as a confocal microscope or a wide-field fluorescence microscope coupled to a charge-coupled device (CCD) camera. It is readily applied to samples prepared with standard immunofluorescence techniques using antibodies labeled with fluorescent dyes that act as a donor and acceptor pair for FRET. Energy transfer efficiencies are quantified based on the release of quenching of donor fluorescence due to FRET, measured by comparing the intensity of donor fluorescence before and after complete photobleaching of the acceptor. As described, this method uses Cy3 and Cy5 as the donor and acceptor fluorophores, but can be adapted for other FRET pairs Including cyan fluorescent protein and yellow fluorescent protein, (C) 2001 Academic Press.