Mazza Davide, Stasevich Timothy J, Karpova Tatiana S, McNally James G
Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
Methods Mol Biol. 2012;833:177-200. doi: 10.1007/978-1-61779-477-3_12.
Live-cell microscopy has demonstrated that many nuclear proteins bind transiently 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.
活细胞显微镜技术已证明，许多核蛋白会短暂地与染色质中的靶位点结合。这些结合相互作用可通过两种相关的活细胞成像技术进行检测和定量，即荧光相关光谱法（FCS）和时间图像相关光谱法（TICS）。通过适当的定量建模，可以从FCS和TICS数据中获得核蛋白与染色质结合和解离速率的估计值。这些结合速率可以计算出细胞核中游离蛋白和结合蛋白的比例，以及从一个结合位点扩散到下一个结合位点所需的时间和在染色质靶标上的停留时间。在本方案中，我们总结了FCS和TICS的基本原理，然后描述了应如何收集和分析这些数据以提取体内结合的估计值。
