Vereb György, Nagy Péter, Szöllosi János
Department of Biophysics and Cell Biology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary.
Methods Mol Biol. 2011;699:371-92. doi: 10.1007/978-1-61737-950-5_18.
Investigation of protein-protein interactions in situ in living or intact cells gains expanding importance as structure/function relationships proposed from bulk biochemistry and molecular modeling experiments require demonstration at the cellular level. Fluorescence resonance energy transfer (FRET)-based methods are excellent tools for determining proximity and supramolecular organization of biomolecules at the cell surface or inside the cell. This could well be the basis for the increasing popularity of FRET; in fact, the number of publications exploiting FRET has doubled in the past 5 years. In this chapter, we intend to provide a generally useable protocol for measuring FRET in flow cytometry. After a concise theoretical introduction, recipes are provided for successful labeling techniques and measurement approaches. The simple, quenching-based population-level measurement; the classic ratiometric, intensity-based technique providing cell-by-cell actual FRET efficiencies, and a more advanced version of the latter, allowing for cell-by-cell autofluorescence correction, are described. Finally, points of caution are given to help design proper experiments and critically interpret the results.
随着基于大量生物化学和分子建模实验提出的结构/功能关系需要在细胞水平上进行验证，对活细胞或完整细胞中蛋白质-蛋白质相互作用的原位研究变得越来越重要。基于荧光共振能量转移（FRET）的方法是确定细胞表面或细胞内生物分子的接近程度和超分子组织的优秀工具。这很可能是FRET越来越受欢迎的基础；事实上，在过去5年中，利用FRET的出版物数量翻了一番。在本章中，我们打算提供一种在流式细胞术中测量FRET的通用可用方案。在进行简要的理论介绍之后，将提供成功的标记技术和测量方法的配方。描述了简单的基于淬灭的群体水平测量；提供逐个细胞实际FRET效率的经典比率法、基于强度的技术，以及后者的更高级版本，该版本允许逐个细胞进行自发荧光校正。最后，给出了注意事项，以帮助设计适当的实验并批判性地解释结果。
