Mahen Robert, Koch Birgit, Wachsmuth Malte, Politi Antonio Z, Perez-Gonzalez Alexis, Mergenthaler Julia, Cai Yin, Ellenberg Jan
European Molecular Biology Laboratory, 69117 Heidelberg, Germany.
European Molecular Biology Laboratory, 69117 Heidelberg, Germany
Mol Biol Cell. 2014 Nov 5;25(22):3610-8. doi: 10.1091/mbc.E14-06-1091. Epub 2014 Sep 17.
Fluorescence tagging of proteins is a widely used tool to study protein function and dynamics in live cells. However, the extent to which different mammalian transgene methods faithfully report on the properties of endogenous proteins has not been studied comparatively. Here we use quantitative live-cell imaging and single-molecule spectroscopy to analyze how different transgene systems affect imaging of the functional properties of the mitotic kinase Aurora B. We show that the transgene method fundamentally influences level and variability of expression and can severely compromise the ability to report on endogenous binding and localization parameters, providing a guide for quantitative imaging studies in mammalian cells.
蛋白质的荧光标记是一种广泛用于研究活细胞中蛋白质功能和动态的工具。然而，不同的哺乳动物转基因方法在多大程度上能够如实反映内源性蛋白质的特性，尚未进行过比较研究。在这里，我们使用定量活细胞成像和单分子光谱来分析不同的转基因系统如何影响有丝分裂激酶Aurora B功能特性的成像。我们表明，转基因方法从根本上影响表达水平和变异性，并可能严重损害报告内源性结合和定位参数的能力，为哺乳动物细胞中的定量成像研究提供了指导。
