GFP-based FRET analysis in live cells
GFP-based FRET analysis in live cells
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DOI：
10.1016/j.brainres.2006.01.119
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发表时间：
2006-05-26
期刊：
影响因子：
2.9
通讯作者：
Zheng, Jie
中科院分区：
文献类型：
--
作者：
Takanishi, Christina L.;Bykova, Ekaterina A.;Zheng, Jie
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 nm的小距离。近年来，由于绿色荧光蛋白（GFP）和许多改进的变体的发现，其应用得到了极大的推广，这些变体构成了良好的供受体荧光团对。基于gfp的蛋白质结构稳定，相对惰性，并且可以可靠地附着在感兴趣的点上。基于gfp的FRET技术的易于访问和它在许多应用中的明显有用性的组合可能导致自满。潜在的问题，如光污染物，如漏血和串扰，以及供体和受体浓度不一致，很容易被忽视，并可能导致FRET计算和数据解释的错误。在本文中，我们概述了基于gfp的FRET可能存在的缺陷以及解决这些问题的方法，包括可以轻松应用于活细胞研究的“光谱FRET”技术。(c) 2006 Elsevier B.V.版权所有
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. (c) 2006 Elsevier B.V. All rights reserved.