Electroporation of Labeled Antibodies to Visualize Endogenous Proteins and Posttranslational Modifications in Living Metazoan Cell Types.
Electroporation of Labeled Antibodies to Visualize Endogenous Proteins and Posttranslational Modifications in Living Metazoan Cell Types.
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DOI：
10.21769/bioprotoc.3069
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发表时间：
2018-11-05
期刊：
影响因子：
0.8
通讯作者：
Weiss E
中科院分区：
文献类型：
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作者：
Conic S;Desplancq D;Tora L;Weiss E
不同细胞内因子的实时时空定位及其在活细胞中的检测是了解基于蛋白质的动态过程的重要参数。因此，需要进行活细胞成像并测量单细胞中的内源蛋白质动态。然而，在不过度表达融合蛋白或基因标签的情况下对活细胞中的内源蛋白进行荧光标记通常是不可能的。在这里，我们描述了一种基于抗体的多功能成像方法（VANIMA），能够精确定位和跟踪活细胞中的内源蛋白。标记是通过将荧光染料偶联的抗体或抗体片段 (Fab) 高效且无害地递送到活细胞中以及这些抗体与细胞内的目标蛋白特异性结合来实现的。我们的方案逐步描述了从测试所需抗体的适用性、将抗体消化到 Fab 直至标记和通过电穿孔将抗体或 Fab 递送到细胞中的过程。 VANIMA 可以适应任何单克隆抗体，无论是自制的还是商业的，以及许多不同的后生动物细胞系。此外，我们的方法易于实现，不仅可以用于可视化和跟踪内源性因素，还可以特异性标记翻译后修饰，这是迄今为止任何其他标记技术无法实现的。
The spatiotemporal localization of different intracellular factors in real-time and their detection in live cells are important parameters to understand dynamic protein-based processes. Therefore, there is a demand to perform live-cell imaging and to measure endogenous protein dynamics in single cells. However, fluorescent labeling of endogenous protein in living cells without overexpression of fusion proteins or genetic tagging has not been routinely possible. Here we describe a versatile antibody-based imaging approach (VANIMA) to be able to precisely locate and track endogenous proteins in living cells. The labeling is achieved by the efficient and harmless delivery of fluorescent dye-conjugated antibodies or antibody fragments (Fabs) into living cells and the specific binding of these antibodies to the target protein inside of the cell. Our protocol describes step by step the procedure from testing of the suitability of the desired antibody, over the digestion of the antibody to Fabs until the labeling and the delivery by electroporation of the antibody or Fab into the cells. VANIMA can be adapted to any monoclonal antibody, self-produced or commercial, and many different metazoan cell lines. Additionally, our method is simple to implement and can be used not only to visualize and track endogenous factors, but also to specifically label posttranslational modifications, which cannot be achieved by any other labeling technique so far.