Venkatesan Shriram, Kandola Tejbir S, Rodríguez-Gama Alejandro, Box Andrew, Halfmann Randal
Stowers Institute for Medical Research.
Stowers Institute for Medical Research; Department of Molecular and Integrative Physiology, The University of Kansas School of Medicine;
J Vis Exp. 2019 Jul 17(149). doi: 10.3791/59577.
Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.
蛋白质自组装决定蛋白质功能，并在空间和时间上对细胞过程进行分隔。目前用于研究它的方法存在灵敏度低、间接读数、通量有限和/或群体水平而非单细胞分辨率等问题。我们设计了一种基于流式细胞术的单一方法来解决所有这些限制：分布式双荧光FRET或DAmFRET。DAmFRET通过体内敏化发射FRET检测和量化蛋白质自组装，能够在从酵母到人类细胞的各种模型系统中应用，并实现灵敏的单细胞高通量读数，而与蛋白质的定位或溶解性无关。
