Department of Chemistry, The Pennsylvania State University, University Park, PA, United States.
Department of Chemistry, The Pennsylvania State University, University Park, PA, United States; Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, United States; The Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA, United States.
Methods Enzymol. 2020;639:1-22. doi: 10.1016/bs.mie.2020.04.006. Epub 2020 Apr 28.
Protein aggregation is a process that occurs through the self-assembly of misfolded proteins to form soluble oligomers and insoluble aggregates. While there has been significant interest in protein aggregation for neurodegenerative diseases, progress in this field of research has been limited by the lack of effective methods to detect and interrogate these species in live cells. To resolve this issue, we have developed a new imaging method named the AggTag to report on protein aggregation in live cells with fluorescence microscopy. The AggTag method utilizes a genetic fusion of a protein of interest (POI) to a protein tag to conjugate with the AggTag probe, which contains a fluorophore that turns on its fluorescence upon interaction with protein aggregates. Unlike the conventional methods, this method enables one to detect soluble misfolded oligomers that were previously invisible. Furthermore, the AggTag method has been applied for the simultaneous detection of co-aggregation between two different POIs by a dual-color and orthogonal tagging system. This chapter aims to provide step-by-step procedures of the AggTag method for researchers who intend to study aggregation of POIs in mammalian cell lines.
蛋白质聚集是一种通过错误折叠的蛋白质自组装形成可溶性寡聚物和不溶性聚集体的过程。虽然人们对神经退行性疾病中的蛋白质聚集产生了浓厚的兴趣，但由于缺乏有效方法来检测和研究活细胞中的这些物质，该领域的研究进展受到了限制。为了解决这个问题，我们开发了一种新的成像方法，名为 AggTag，用于通过荧光显微镜报告活细胞中的蛋白质聚集。AggTag 方法利用感兴趣的蛋白质（POI）与蛋白质标签的基因融合，与 AggTag 探针缀合，该探针包含一个荧光团，当与蛋白质聚集体相互作用时，其荧光会开启。与传统方法不同，该方法可以检测到以前不可见的可溶性错误折叠的寡聚物。此外，AggTag 方法已通过双色和正交标记系统应用于同时检测两种不同 POI 之间的共聚集。本章旨在为计划在哺乳动物细胞系中研究 POI 聚集的研究人员提供 AggTag 方法的分步程序。
