Suppr超能文献

通过光漂白后的荧光恢复分析结合反应。

Analysis of binding reactions by fluorescence recovery after photobleaching.

作者信息

Sprague Brian L, Pego Robert L, Stavreva Diana A, McNally James G

机构信息

Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.

出版信息

Biophys J. 2004 Jun;86(6):3473-95. doi: 10.1529/biophysj.103.026765.

Abstract

Fluorescence recovery after photobleaching (FRAP) is now widely used to investigate binding interactions in live cells. Although various idealized solutions have been identified for the reaction-diffusion equations that govern FRAP, there has been no comprehensive analysis or systematic approach to serve as a guide for extracting binding information from an arbitrary FRAP curve. Here we present a complete solution to the FRAP reaction-diffusion equations for either single or multiple independent binding interactions, and then relate our solution to the various idealized cases. This yields a coherent approach to extract binding information from FRAP data which we have applied to the question of transcription factor mobility in the nucleus. We show that within the nucleus, the glucocorticoid receptor is transiently bound to a single state, with each molecule binding on average 65 sites per second. This rapid sampling is likely to be important in finding a specific promoter target sequence. Further we show that this predominant binding state is not the nuclear matrix, as some studies have suggested. We illustrate how our analysis provides several self-consistency checks on a FRAP fit. We also define constraints on what can be estimated from FRAP data, show that diffusion should play a key role in many FRAP recoveries, and provide tools to test its contribution. Overall our approach establishes a more general framework to assess the role of diffusion, the number of binding states, and the binding constants underlying a FRAP recovery.

摘要

光漂白后荧光恢复(FRAP)现已广泛用于研究活细胞中的结合相互作用。尽管已经为控制FRAP的反应扩散方程确定了各种理想化的解决方案,但尚未有全面的分析或系统的方法来指导从任意FRAP曲线中提取结合信息。在此,我们给出了针对单种或多种独立结合相互作用的FRAP反应扩散方程的完整解决方案,然后将我们的解决方案与各种理想化情况联系起来。这产生了一种从FRAP数据中提取结合信息的连贯方法,我们已将其应用于细胞核中转录因子迁移率的问题。我们表明,在细胞核内,糖皮质激素受体短暂地结合到单一状态,每个分子平均每秒结合65个位点。这种快速采样可能对于找到特定的启动子靶序列很重要。此外,我们表明这种主要的结合状态并非如一些研究所暗示的那样是核基质。我们说明了我们的分析如何对FRAP拟合提供几种自洽性检验。我们还定义了从FRAP数据中可以估计的内容的限制,表明扩散在许多FRAP恢复中应起关键作用,并提供了测试其贡献的工具。总体而言,我们的方法建立了一个更通用的框架,以评估扩散的作用、结合状态的数量以及FRAP恢复背后的结合常数。

文献AI研究员

20分钟写一篇综述,助力文献阅读效率提升50倍。

立即体验

用中文搜PubMed

大模型驱动的PubMed中文搜索引擎

马上搜索

文档翻译

学术文献翻译模型,支持多种主流文档格式。

立即体验