Karpova Tatiana, McNally James G
National Institutes of Health, Bethesda, Maryland, USA.
Curr Protoc Cytom. 2006 Feb;Chapter 12:Unit12.7. doi: 10.1002/0471142956.cy1207s35.
FRET is a light microscopy method for detecting protein-protein interactions within intact cells. The FRET protocol presented here is for CFP- and YFP-tagged proteins examined with an argon laser on a scanning confocal microscope. FRET is assayed by one of the most straightforward approaches available, namely, acceptor photobleaching. In this procedure, the YFP-tagged protein (the FRET "acceptor") is photobleached at a cellular site of interest, and then the intensity of the CFP-tagged protein (the FRET "donor") at that same site is measured. In principle, FRET is detected when the CFP intensity increases after the photobleaching of YFP. This unit describes the appropriate steps to perform this measurement, as well as the necessary controls to ensure that an increase in CFP intensity, if detected, in fact reflects bona fide FRET. Successful application of the protocol will support the conclusion that the CFP- and YFP-tagged proteins directly interact at the site of the photobleaching.
荧光共振能量转移（FRET）是一种用于检测完整细胞内蛋白质-蛋白质相互作用的光学显微镜方法。此处介绍的FRET实验方案适用于在扫描共聚焦显微镜上用氩离子激光检测的CFP（青色荧光蛋白）和YFP（黄色荧光蛋白）标记的蛋白质。FRET通过一种最直接的可用方法进行检测，即受体光漂白。在该过程中，YFP标记的蛋白质（FRET“受体”）在感兴趣的细胞位点被光漂白，然后测量同一位置CFP标记的蛋白质（FRET“供体”）的强度。原则上，当YFP光漂白后CFP强度增加时，可检测到FRET。本单元描述了进行该测量的适当步骤，以及必要的对照，以确保如果检测到CFP强度增加，实际上反映的是真正的FRET。该实验方案的成功应用将支持CFP和YFP标记的蛋白质在光漂白位点直接相互作用的结论。
