| Combining Gel Retardation and Footprinting to Determine Protein-DNA Interactions of Specific and/or Less Stable Complexes
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Author:
Date:
2020-12-05
[Abstract] DNA footprinting is a classic technique to investigate protein-DNA interactions. However, traditional footprinting protocols can be unsuccessful or difficult to interpret if the binding of the protein to the DNA is weak, the protein has a fast off-rate, or if several different protein-DNA complexes are formed. Our protocol differs from traditional footprinting protocols, because it provides a method to isolate the protein-DNA complex from a native gel after treatment with the footprinting agent, thus removing the bound DNA from the free DNA or other protein-DNA complexes. The DNA is then extracted from the isolated complex before electrophoresis on a sequencing gel to determine the footprinting pattern. This analysis provides a possible solution for those who have been unable to use ...
[摘要] [摘要] DNA足迹是研究蛋白质-DNA相互作用的经典技术。但是，如果蛋白质与DNA的结合较弱，蛋白质的脱落速率较快，或者形成了几种不同的蛋白质-DNA复合物，则传统的足迹方案可能会失败或难以解释。我们的协议不同于传统的足迹协议，因为它提供了一种在使用足迹剂处理后从天然凝胶中分离蛋白质-DNA复合物的方法，从而从游离DNA或其他蛋白质-DNA复合物中去除了结合的DNA。然后从分离的复合物中提取DNA，然后在测序凝胶上电泳以确定印迹模式。该分析为无法使用传统足迹法确定蛋白质与DNA接触的人提供了可能的解决方案。
[背景]核酸酶/化学足迹是一个典型的方法来探测蛋白质-DNA相互作用（腊士和施米茨，1978;萨瑟-德怀特和Gralla，1989;汉普等人，2007） ...
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| Electrophoresis Mobility Shift Assay
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Author:
Date:
2014-04-05
[Abstract] Protein (transcription factors and/or transcription cofactors)-binding to DNA is a critical event in regulation of transcription. Electrophoresis Mobility Shift Assay (EMSA), also known as gel shift assay, is a useful tool to detect protein- or protein complex-DNA/RNA interaction and to evaluate DNA binding specificity of transcription factors in vitro. Here we describe a simple method for EMSA with fluorescent dye-bound oligo DNA probes and recombinant protein expressed in bacterial cells. Using fluorescent dye instead of radioisotope enables easy handling and long-term storage of labelled-probes without reduction of detection sensitivity.
[摘要] 与DNA结合的蛋白质（转录因子和/或转录辅因子）是调节转录的关键事件。 电泳迁移率变动分析（EMSA），也称为凝胶移位分析，是检测蛋白质或蛋白质复合物-DNA/RNA相互作用和评价转录因子的体外DNA结合特异性的有用工具。 在这里我们介绍一种简单的方法与荧光染料绑定寡DNA探针和表达在细菌细胞中的重组蛋白的EMSA。 使用荧光染料代替放射性同位素使得容易处理和长期储存标记的探针而不降低检测灵敏度。
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| EMSA Analysis of DNA Binding By Rgg Proteins
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Author:
Date:
2013-08-05
[Abstract] In bacteria, interaction of various proteins with DNA is essential for the regulation of specific target gene expression. Electrophoretic mobility shift assay (EMSA) is an in vitro approach allowing for the visualization of these protein-DNA interactions. Rgg proteins comprise a family of transcriptional regulators widespread among the Firmicutes. Some of these proteins function independently to regulate target gene expression, while others have now been demonstrated to function as effectors of cell-to-cell communication, having regulatory activitiesthat that are modulated via direct interaction with small signaling peptides. EMSA analysis can be used to assess DNA binding of either type of Rgg protein. EMSA analysis of Rgg protein activity has facilitated in vitro ...
[摘要] 在细菌中，各种蛋白质与DNA的相互作用对于调节特异性靶基因表达是必需的。电泳迁移率变动分析（EMSA）是允许这些蛋白质-DNA相互作用的可视化的体外方法。 Rgg蛋白包括在坚牢菌中广泛的转录调节子家族。这些蛋白质中的一些独立地起作用以调节靶基因表达，而其它蛋白质已经被证明作为细胞与细胞间通信的效应物起作用，具有通过与小信号肽的直接相互作用调节的调节活性。 EMSA分析可用于评估任一类型的Rgg蛋白的DNA结合。 EMSG对Rgg蛋白活性的分析促进了体外证实调节靶标，通过DNA探针诱变鉴定精确的DNA结合位点，以及表征一些同源信号肽调节Rgg蛋白功能的机制（<例如在某些情况下中断DNA结合）。
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