A flow cytometry-based method to simplify the analysis and quantification of protein association to chromatin in mammalian cells.
A flow cytometry-based method to simplify the analysis and quantification of protein association to chromatin in mammalian cells.
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DOI：
10.1038/nprot.2015.066
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发表时间：
2015-09
期刊：
影响因子：
14.8
通讯作者：
Jackson SP
中科院分区：
文献类型：
--
作者：
Forment JV;Jackson SP
染色质上的蛋白质积累传统上使用免疫荧光显微镜或生化细胞分级分离，然后进行Western免疫印迹分析来研究。为了提高这种分析的重现性，使其更容易量化，并允许在高通量筛选中的流线型应用，我们最近将经典的免疫荧光显微镜检测技术与流式细胞术相结合。除了上述特征之外，通过将其与DNA含量和DNA复制的检测相结合，该方法允许明确和直接地将蛋白质缔合的细胞周期分布分配给染色质，而不需要细胞培养同步化。此外，它相对快速（从样品收集到定量不超过一个工作日），与标准生物化学分级方法相比需要更少的起始材料，并且克服了免疫荧光显微镜所需的扁平粘附细胞类型的需要。
Protein accumulation on chromatin has traditionally been studied using immunofluorescence microscopy or biochemical cellular fractionation followed by western immunoblot analysis. As a way to improve the reproducibility of this kind of analysis, make it easier to quantify and allow a stream-lined application in high-throughput screens, we recently combined a classical immunofluorescence microscopy detection technique with flow cytometry. In addition to the features described above, and by combining it with detection of both DNA content and DNA replication, this method allows unequivocal and direct assignment of cell-cycle distribution of protein association to chromatin without the need for cell culture synchronization. Furthermore, it is relatively quick (no more than a working day from sample collection to quantification), requires less starting material compared to standard biochemical fractionation methods and overcomes the need for flat, adherent cell types that are required for immunofluorescence microscopy.