光漂白后的荧光恢复
光漂白
动力学
生物信息学
荧光显微镜
荧光
生物系统
生物物理学
故障排除
显微镜
化学
计算机科学
生物
物理
生物化学
光学
量子力学
基因
操作系统
作者
Nickolaos Nikiforos Giakoumakis,Maria Anna Rapsomaniki,Zoi Lygerou
出处
期刊:Methods in molecular biology
日期:2017-01-01
卷期号:: 243-267
被引量:22
标识
DOI:10.1007/978-1-4939-6810-7_16
摘要
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.
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