Quantitative Analysis of Protein-DNA Interaction by qDPI-ELISA
Quantitative Analysis of Protein-DNA Interaction by qDPI-ELISA
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DOI：
10.1007/978-1-4939-6396-6_4
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发表时间：
2016-01-01
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影响因子：
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通讯作者：
Wanke, Dierk
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文献类型：
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作者：
Fischer, Stefan M.;Boeser, Alexander;Wanke, Dierk
DNA 结合蛋白与其同源 DNA 基序的特异性结合是体内基因表达控制和染色质组织的关键步骤。开发用于鉴定体内结合区域的方法，例如通过染色质免疫沉淀 (ChIP) 或 DNA 腺嘌呤甲基转移酶鉴定 (Dam-ID) 为系统生物学中的数据挖掘或合成生物学中的应用添加了额外级别的定性信息。在这方面，体内技术超越了全面表征蛋白质-DNA 相互作用的方法，特别是单碱基对分辨率的结合基序。蛋白质的 DNA 结合能力的阐明通常使用酵母单杂交、电泳迁移率变动测定 (EMSA) 或指数富集配体系统进化 (SELEX) 等方法来完成，这些方法仅提供定性结合信息，不适用于多个 DNA 基序的自动化或高通量筛选。在这里，我们描述了定量 DNA-蛋白质相互作用-ELISA (qDPI-ELISA) 方案，该方案利用荧光融合蛋白，因此比经典的 DPI-ELISA 更快、更容易处理。尽管每个 DPI-ELISA 实验都能提供定量信息，但 qDPI-ELISA 的一致性更高，因为它不依赖于免疫学检测。我们在 qDPI-ELISA 实验中证明了探针和不同蛋白质提取物之间的高度可比性。
The specific binding of DNA-binding proteins to their cognate DNA motifs is a crucial step for gene expression control and chromatin organization in vivo. The development of methods for the identification of in vivo binding regions by, e.g. chromatin immunoprecipitation (ChIP) or DNA adenine methyltransferase identification (Dam-ID) added an additional level of qualitative information for data mining in systems biology or applications in synthetic biology. In this respect, the in vivo techniques outpaced methods for thorough characterization of protein-DNA interaction and, especially, of the binding motifs at single base-pair resolution. The elucidation of DNA-binding capacities of proteins is frequently done with methods such as yeast one-hybrid, electrophoretic mobility shift assay (EMSA) or systematic evolution of ligands by exponential enrichment (SELEX) that provide only qualitative binding information and are not suited for automation or high-throughput screening of several DNA motifs. Here, we describe the quantitative DNA-protein-Interaction-ELISA (qDPI-ELISA) protocol, which makes use of fluorescent fusion proteins and, hence, is faster and easier to handle than the classical DPI-ELISA. Although every DPI-ELISA experiment delivers quantitative information, the qDPI-ELISA has an increased consistency, as it does not depend on immunological detection. We demonstrate the high comparability between probes and different protein extracts in qDPI-ELISA experiments.