Dhar Apratim, Gruebele Martin
University of Illinois, Urbana, Illinois, USA.
Curr Protoc Protein Sci. 2011 Aug;Chapter 28:Unit28.1. doi: 10.1002/0471140864.ps2801s65.
This protocol describes the technique of Fast Relaxation Imaging (FReI) as applied to protein folding inside living cells. The required modifications of a fluorescence microscope by addition of a diode laser temperature jump source, a yellow/blue switchable light-emitting diode source, and a two-color CCD camera to collect movies of protein dynamics inside cells are discussed. A description of how proteins are labeled for imaging, how cells are prepared for imaging, and how imaging of kinetics inside cells with millisecond time resolution is obtained, along with the complementary in vitro experiments, is also provided. The ability to carry out comparative in vitro and "in-cell" measurements on the same setup allows for direct comparison of the features distinguishing cellular protein folding (or other biomolecular processes) from studies performed in dilute solution.
本方案描述了应用于活细胞内蛋白质折叠的快速弛豫成像（FReI）技术。讨论了通过添加二极管激光温度跃变源、黄/蓝可切换发光二极管源和双色电荷耦合器件相机对荧光显微镜进行所需的改装，以收集细胞内蛋白质动力学的影像。还提供了关于如何对蛋白质进行标记以用于成像、如何制备细胞用于成像以及如何以毫秒时间分辨率获得细胞内动力学成像的描述，以及补充的体外实验。在同一装置上进行体外和“细胞内”比较测量的能力，使得能够直接比较区分细胞内蛋白质折叠（或其他生物分子过程）与在稀溶液中进行的研究的特征。
