Takanishi Christina L, Bykova Ekaterina A, Cheng Wei, Zheng Jie
Department of Physiology and Membrane Biology, University of California School of Medicine, One Shields Avenue, Davis, CA 95616, USA.
Brain Res. 2006 May 26;1091(1):132-9. doi: 10.1016/j.brainres.2006.01.119. Epub 2006 Mar 10.
Fluorescence resonance energy transfer (FRET) is a widely utilized optical technique for measuring small distances of 1-10 nm in live cells. In recent years, its application has been greatly popularized by the discovery of green fluorescent protein (GFP) and many improved variants which make good donor-acceptor fluorophore pairs. GFP-based proteins are structurally stable, relatively inert, and can be reliably attached to points of interest. The combination of easy access to the GFP-based FRET technique and its obvious usefulness in many applications can lead to complacency. Potential problems such as light contaminants, e.g., bleed-through and cross-talk, and inconsistent donor and acceptor concentrations are easily overlooked and can lead to errors in FRET calculation and data interpretation. In this article, we outline possible pitfalls of GFP-based FRET and approaches that address these issues, including a "Spectra FRET" technique that can be easily applied to live cell studies.
荧光共振能量转移（FRET）是一种广泛应用的光学技术，用于测量活细胞中1-10纳米的微小距离。近年来，绿色荧光蛋白（GFP）及其许多改进变体的发现极大地推广了其应用，这些变体构成了良好的供体-受体荧光团对。基于GFP的蛋白质结构稳定，相对惰性，并且可以可靠地连接到感兴趣的位点。基于GFP的FRET技术易于获得，且在许多应用中具有明显的实用性，这可能导致自满。诸如光污染物（例如渗漏和串扰）以及供体和受体浓度不一致等潜在问题很容易被忽视，并且可能导致FRET计算和数据解释中的错误。在本文中，我们概述了基于GFP的FRET可能存在的陷阱以及解决这些问题的方法，包括一种可以轻松应用于活细胞研究的“光谱FRET”技术。
