Novel fluorescence labeling and high-throughput assay technologies for in vitro analysis of protein interactions
Novel fluorescence labeling and high-throughput assay technologies for in vitro analysis of protein interactions
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DOI：
10.1101/gr.218802
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发表时间：
2002-03-01
期刊：
影响因子：
7
通讯作者：
Yanagawa, H
中科院分区：
文献类型：
--
作者：
Doi, N;Takashima, H;Yanagawa, H
我们开发并测试了一种简单的方法，用于基于高效的体外翻译系统结合高通量技术（如微阵列和荧光互相关光谱（FCCS））的蛋白质荧光标记和相互作用分析。通过使用通过脱氧胞苷酸接头与各种荧光团连接的嘌呤霉素类似物，在体外翻译期间，单个荧光团可以有效地掺入蛋白质的羧基末端。我们证实，所得到的荧光标记的蛋白质是有用的探测蛋白质-蛋白质和蛋白质-DNA相互作用的pulldown测定，DNA微阵列，和FCCS在模型实验。这些荧光分析系统可以很容易地扩展到功能基因组学研究中的蛋白质相互作用的高度并行分析。
We developed and tested a simple method for fluorescence labeling and interaction analysis of proteins based on a highly efficient in vitro translation system combined with high-throughput technologies such as microarrays and fluorescence cross-correlation spectroscopy (FCCS). By use of puromycin analogs linked to various fluorophores through a deoxycytidylic acid linker, a single fluorophore can be efficiently incorporated into a protein at the carboxyl terminus during in vitro translation. We confirmed that the resulting fluorescently labeled proteins are useful for probing protein-protein and protein-DNA interactions by means of pulldown assay, DNA microarrays, and FCCS in model experiments. These fluorescence assay systems can be easily extended to highly parallel analysis of protein interactions in studies of functional genomics.