Monitoring dynamic binding of chromatin proteins in vivo by fluorescence correlation spectroscopy and temporal image correlation spectroscopy.
Monitoring dynamic binding of chromatin proteins in vivo by fluorescence correlation spectroscopy and temporal image correlation spectroscopy.
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DOI：
10.1007/978-1-61779-477-3_12
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
McNally JG
中科院分区：
文献类型：
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作者：
Mazza D;Stasevich TJ;Karpova TS;McNally JG
活细胞显微镜已证明许多核蛋白短暂结合到染色质的靶位点。这些结合相互作用可以通过两种相关的活细胞成像技术荧光相关光谱（FCS）和时间图像相关光谱（TICS）来检测和量化。通过适当的定量模型，可以从 FCS 和 TICS 数据中获得核蛋白与染色质结合的结合率和解离率的估计值。这些结合率允许计算细胞核中游离和结合蛋白质的分数，加上从一个结合位点扩散到下一个结合位点所需的时间以及在染色质靶标上的停留时间。在本协议中，我们总结了 FCS 和 TICS 的基本原理，然后描述如何收集和分析这些数据以提取体内结合的估计值。
Live-cell microscopy has demonstrated that many nuclearproteins bind trransiently to target sites in chromatin. These binding interactions can be detected and quantified by two related live-cell imaging techniques, Fluorescence Correlation Spectroscopy (FCS) and Temporal Image Correlation Spectroscopy (TICS). With proper quantitative modeling, it is possible to obtain estimates from FCS and TICS data of the association and dissociation rates of nuclear protein binding to chromatin. These binding rates permit calculating the fractions of free and bound protein in the nucleus, plus the time required to diffuse from one binding site to the next and the dwell time on a chromatin target. In this protocol, we summarize the underlying principles of FCS and TICS, and then describe how these data should be collected and analyzed to extract estimates of in vivo binding.