Nomoto Mika, Tada Yasuomi, Tsukagoshi Hironaka
Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan.
Center for Gene Research, Nagoya University, Nagoya, Japan.
Bio Protoc. 2019 Feb 5;9(3):e3155. doi: 10.21769/BioProtoc.3155.
Identification of specific DNA binding sites of transcription factors is important in understanding their functions. Recent techniques allow us to investigate genome-wide binding positions by chromatin immunoprecipitation combined with high-throughput sequencing. However, to further explore the binding motifs of transcription factors, in-depth biochemical analysis is required. Here, we describe an efficient protocol of protein-DNA interactions based on a combination of our transcription/translation system and AlphaScreen technology. The transcription/translation system supports an efficient and quick way of protein synthesis by alleviating cumbersome cloning steps. In addition, AlphaScreen system provides a highly sensitive, quick, and easy handling platform to investigate the protein-DNA interactions . Thus, our method largely contributes to comprehensive analysis of the biochemical properties of transcription factors.
鉴定转录因子的特定DNA结合位点对于理解其功能至关重要。最近的技术使我们能够通过染色质免疫沉淀结合高通量测序来研究全基因组的结合位置。然而，为了进一步探索转录因子的结合基序，需要进行深入的生化分析。在此，我们描述了一种基于我们的转录/翻译系统与AlphaScreen技术相结合的蛋白质-DNA相互作用的高效方案。转录/翻译系统通过省去繁琐的克隆步骤，支持一种高效快速的蛋白质合成方式。此外，AlphaScreen系统为研究蛋白质-DNA相互作用提供了一个高度灵敏、快速且易于操作的平台。因此，我们的方法在很大程度上有助于对转录因子的生化特性进行全面分析。
