使用APEX标记的条件稳定纳米抗体对哺乳动物细胞中的蛋白质和蛋白质复合物进行电子显微镜检测。
Electron Microscopic Detection of Proteins and Protein Complexes in Mammalian Cells Using APEX-tagged, Conditionally Stable Nanobodies.
作者信息
Hall Thomas E, Rae James, Ariotti Nicholas, G Parton Robert
机构信息
The University of Queensland, Institute for Molecular Bioscience, Queensland 4072, Australia.
The University of Queensland, Centre for Microscopy and Microanalysis, Brisbane, Queensland 4072, Australia.
出版信息
Bio Protoc. 2018 Nov 20;8(22):e3088. doi: 10.21769/BioProtoc.3088.
The recent development of 3D electron microscopic techniques for cells and tissues has necessitated the development of new methods for the detection of proteins and protein-complexes . The development of new genetic tags, such as the ascorbate peroxidase, APEX, for electron microscopic detection of tagged proteins has expanded the available toolbox and ushered in a new era in biological electron microscopy. Here, we describe methods for combining conditionally-stable nanobodies to fluorescent protein tags with APEX-based detection. These methods are compatible with detection of low levels of expression of fluorescently-tagged proteins and with detection of protein complexes using split GFP-based complementation methods. We describe a simple protocol for applying these methods to the electron microscopic detection of proteins and protein complexes in cultured cells.
用于细胞和组织的3D电子显微镜技术的最新发展，使得开发检测蛋白质和蛋白质复合物的新方法成为必要。新的基因标签的开发，如用于电子显微镜检测标记蛋白的抗坏血酸过氧化物酶APEX，扩展了可用的工具箱，并开创了生物电子显微镜的新纪元。在这里，我们描述了将条件稳定的纳米抗体与荧光蛋白标签结合用于基于APEX检测的方法。这些方法与检测荧光标记蛋白的低水平表达以及使用基于分裂GFP的互补方法检测蛋白质复合物兼容。我们描述了一个简单的方案，用于将这些方法应用于培养细胞中蛋白质和蛋白质复合物的电子显微镜检测。
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