Feinstein Timothy N
Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA, USA.
Methods Mol Biol. 2013;1066:121-9. doi: 10.1007/978-1-62703-604-7_11.
Förster resonance energy transfer (FRET) is a proximity-dependent quantum effect that allows the measurement of protein interactions and conformational changes which are invisible to traditional forms of fluorescence or electron microscopy. However, FRET experiments often have difficulty detecting interactions that are transient and localized or occur in low abundance against a large background. This protocol describes a method of improving on the sensitivity and quantifiability of FRET experiments by using time-specific detection to isolate FRET-mediated acceptor emission from cross-talk excitation and all other sources of nonspecific fluorescence background.
荧光共振能量转移（FRET）是一种依赖于距离的量子效应，它能够测量蛋白质相互作用和构象变化，而这些变化对于传统形式的荧光或电子显微镜来说是不可见的。然而，FRET实验在检测瞬态、局部发生或在大量背景下低丰度出现的相互作用时往往存在困难。本方案描述了一种通过使用时间特异性检测从串扰激发和所有其他非特异性荧光背景源中分离FRET介导的受体发射，从而提高FRET实验的灵敏度和可量化性的方法。
