Giakoumakis Nickolaos Nikiforos, Rapsomaniki Maria Anna, Lygerou Zoi
Laboratory of Biology, School of Medicine, University of Patras, GR26500 Rio, Patras, Greece.
IBM Research Zurich, Säumerstrasse 4, CH-8803, Rüschlikon, Switzerland.
Methods Mol Biol. 2017;1563:243-267. doi: 10.1007/978-1-4939-6810-7_16.
Fluorescence recovery after photobleaching (FRAP) is a cutting-edge live-cell functional imaging technique that enables the exploration of protein dynamics in individual cells and thus permits the elucidation of protein mobility, function, and interactions at a single-cell level. During a typical FRAP experiment, fluorescent molecules in a defined region of interest within the cell are bleached by a short and powerful laser pulse, while the recovery of the fluorescence in the region is monitored over time by time-lapse microscopy. FRAP experimental setup and image acquisition involve a number of steps that need to be carefully executed to avoid technical artifacts. Equally important is the subsequent computational analysis of FRAP raw data, to derive quantitative information on protein diffusion and binding parameters. Here we present an integrated in vivo and in silico protocol for the analysis of protein kinetics using FRAP. We focus on the most commonly encountered challenges and technical or computational pitfalls and their troubleshooting so that valid and robust insight into protein dynamics within living cells is gained.
光漂白后荧光恢复（FRAP）是一种前沿的活细胞功能成像技术，它能够探索单个细胞中的蛋白质动力学，从而在单细胞水平上阐明蛋白质的流动性、功能和相互作用。在典型的FRAP实验中，细胞内特定感兴趣区域的荧光分子被一个短而强的激光脉冲漂白，同时通过延时显微镜随时间监测该区域荧光的恢复情况。FRAP实验设置和图像采集涉及多个步骤，需要仔细执行以避免技术假象。同样重要的是对FRAP原始数据的后续计算分析，以获取关于蛋白质扩散和结合参数的定量信息。在这里，我们展示了一种使用FRAP分析蛋白质动力学的体内和计算机相结合的综合方案。我们关注最常见的挑战、技术或计算陷阱及其故障排除方法，以便获得对活细胞内蛋白质动力学的有效和可靠的见解。
