Llères David, Swift Samuel, Lamond Angus I
Wellcome Trust Biocentre, College of Life Sciences, University of Dundee, United Kingdom.
Curr Protoc Cytom. 2007 Oct;Chapter 12:Unit12.10. doi: 10.1002/0471142956.cy1210s42.
Protein interactions are critical for many processes in mammalian cells. Such interactions include the stable association of proteins within multi-subunit complexes and the transient association of regulatory proteins. Information about protein interactions in cells has previously come from either in vitro analyses using recombinant expressed proteins, or from yeast 2-hybrid studies. A limitation of this approach is that the protein interaction is studied in isolation, without regard to the many competing protein interactions that can occur within cells. This unit presents a light microscopy approach for detecting protein-protein interactions in vivo based on the measurement of FRET using the multiphoton fluorescence lifetime imaging microscopy (FLIM) technique. By using the FLIM-FRET technique, the spatial organization and quantification of such interactions in a living cell can be characterized. A detailed protocol describing the complete microscope procedure and the choice of the appropriate experimental controls as well as the FRET calculations is also included.
蛋白质相互作用对哺乳动物细胞中的许多过程至关重要。此类相互作用包括多亚基复合物中蛋白质的稳定缔合以及调节蛋白的瞬时缔合。此前，有关细胞中蛋白质相互作用的信息要么来自使用重组表达蛋白的体外分析，要么来自酵母双杂交研究。这种方法的一个局限性在于，蛋白质相互作用是孤立研究的，而没有考虑细胞内可能发生的许多竞争性蛋白质相互作用。本单元介绍了一种基于多光子荧光寿命成像显微镜（FLIM）技术测量荧光共振能量转移（FRET）来在体内检测蛋白质-蛋白质相互作用的光学显微镜方法。通过使用FLIM-FRET技术，可以对活细胞中此类相互作用的空间组织和定量进行表征。还包括一份详细的方案，描述了完整的显微镜操作程序、适当实验对照的选择以及FRET计算。
