• 主题
高级搜索  >
历史搜索 清空历史
首页> 美国卫生研究院文献>Scientific Reports >Advanced FRET normalization allows quantitative analysis of protein interactions including stoichiometries and relative affinities in living cells
【2h】

Advanced FRET normalization allows quantitative analysis of protein interactions including stoichiometries and relative affinities in living cells

机译：先进的FRET归一化可定量分析蛋白质相互作用包括活细胞中的化学计量和相对亲和力

代理获取
本网站仅为用户提供外文OA文献查询和代理获取服务，本网站没有原文。下单后我们将采用程序或人工为您竭诚获取高质量的原文，但由于OA文献来源多样且变更频繁，仍可能出现获取不到、文献不完整或与标题不符等情况，如果获取不到我们将提供退款服务。请知悉。
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

FRET (Fluorescence Resonance Energy Transfer) measurements are commonly applied to proof protein-protein interactions. However, standard methods of live cell FRET microscopy and signal normalization only allow a principle assessment of mutual binding and are unable to deduce quantitative information of the interaction. We present an evaluation and normalization procedure for 3-filter FRET measurements, which reflects the process of complex formation by plotting FRET-saturation curves. The advantage of this approach relative to traditional signal normalizations is demonstrated by mathematical simulations. Thereby, we also identify the contribution of critical parameters such as the total amount of donor and acceptor molecules and their molar ratio. When combined with a fitting procedure, this normalization facilitates the extraction of key properties of protein complexes such as the interaction stoichiometry or the apparent affinity of the binding partners. Finally, the feasibility of our method is verified by investigating three exemplary protein complexes. Altogether, our approach offers a novel method for a quantitative analysis of protein interactions by 3-filter FRET microscopy, as well as flow cytometry. To facilitate the application of this method, we created macros and routines for the programs ImageJ, R and MS-Excel, which we make publicly available.
机译：FRET（荧光共振能量转移）测量通常用于证明蛋白质-蛋白质相互作用。但是，活细胞FRET显微镜和信号归一化的标准方法仅允许对相互结合进行原理评估，而无法推断相互作用的定量信息。我们提出了一种针对3滤波器FRET测量的评估和规范化程序，该程序通过绘制FRET饱和曲线来反映复合物形成的过程。数学仿真证明了这种方法相对于传统信号归一化的优势。因此，我们还确定了关键参数的贡献，例如供体和受体分子的总量及其摩尔比。当与拟合过程结合时，这种归一化有助于提取蛋白质复合物的关键特性，例如相互作用的化学计量或结合伴侣的表观亲和力。最后，通过研究三种示例性蛋白质复合物，验证了我们方法的可行性。总之，我们的方法为通过3滤器FRET显微镜以及流式细胞术对蛋白质相互作用进行定量分析提供了一种新颖的方法。为了促进该方法的应用，我们为ImageJ，R和MS-Excel程序创建了宏和例程，并将它们公开提供。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
代理获取

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号