Horizontal Agarose Gel Mobility Shift Assay for Protein-RNA Complexes.
Horizontal Agarose Gel Mobility Shift Assay for Protein-RNA Complexes.
复制标题
蛋白质-RNA 复合物的水平琼脂糖凝胶迁移率变化测定。
DOI：
10.1007/978-1-4939-8793-1_31
复制
发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Lewis,KarenA
中科院分区：
文献类型：
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作者：
Ream,JenniferA;Lewis,LKevin;Lewis,KarenA
琼脂糖凝胶电泳方案的最新进展建立了使用更高电压结合短运行时间的DNA和RNA高分辨率分离的条件。我们随后开发了一种使用这些条件来测量蛋白质对琼脂糖凝胶上的RNA配体的结合亲和力的方案。这种天然凝胶迁移率变动测定非常容易使用，使用大多数实验室中常见的分子生物学试剂。在这里，我们描述了进行天然琼脂糖凝胶电泳的协议，以表征的RNA配体的蛋白质的结合亲和力。电泳时间小于10 min，使蛋白质和配体的解离最小化。我们已经使用p19 siRNA结合蛋白及其同源dsRNA配体来证明用于鉴定最佳条件的策略，以使用该琼脂糖凝胶移位系统来测量表观结合常数。
Recent advances in agarose gel electrophoresis protocols established conditions for the high-resolution separation of DNA and RNA using higher voltages combined with short run times. We subsequently developed a protocol for using these conditions to measure the binding affinity of a protein for an RNA ligand on an agarose gel. This native gel mobility shift assay is highly accessible, using common molecular biology reagents found in most laboratories. Here, we describe the protocol for carrying out native agarose gel electrophoresis to characterize the binding affinity of a protein for an RNA ligand. The electrophoresis time is less than 10 min, which minimizes the dissociation of protein and ligand. We have used the p19 siRNA binding protein and its cognate dsRNA ligand to demonstrate strategies for identifying optimal conditions to measure apparent binding constants using this agarose gel shift system.