Paul Maarten W, Zelensky Alex N, Wyman Claire, Kanaar Roland
Department of Molecular Genetics, Cancer Genomics Center Netherlands, Erasmus University Medical Center, Rotterdam, The Netherlands.
Department of Molecular Genetics, Cancer Genomics Center Netherlands, Erasmus University Medical Center, Rotterdam, The Netherlands; Department of Radiation Oncology, Erasmus University Medical Center, Rotterdam, The Netherlands.
Methods Enzymol. 2018;600:375-406. doi: 10.1016/bs.mie.2017.11.015. Epub 2018 Feb 1.
Direct observation of individual protein molecules in their native environment, at nanometer resolution, in a living cell, in motion is not only fascinating but also uniquely informative. Several recent major technological advances in genomic engineering, protein and synthetic fluorophore development, and light microscopy have dramatically increased the accessibility of this approach. This chapter describes the procedures for modifying endogenous genomic loci to producing fluorescently tagged proteins, their high-resolution visualization, and analysis of their dynamics in mammalian cells, using DNA repair proteins BRCA2 and RAD51 as an example.
在活细胞中，以纳米分辨率直接观察处于天然环境中的单个蛋白质分子，且该分子处于运动状态，这不仅引人入胜，而且具有独特的信息价值。基因组工程、蛋白质和合成荧光团开发以及光学显微镜等领域最近的几项重大技术进步极大地提高了这种方法的可及性。本章以DNA修复蛋白BRCA2和RAD51为例，描述了修饰内源性基因组位点以产生荧光标记蛋白的程序、它们的高分辨率可视化以及在哺乳动物细胞中对其动力学的分析。
