用于蛋白质-RNA复合物的水平琼脂糖凝胶迁移率变动分析
Horizontal Agarose Gel Mobility Shift Assay for Protein-RNA Complexes.
作者信息
Ream Jennifer A, Lewis L Kevin, Lewis Karen A
机构信息
Department of Chemistry and Biochemistry, Texas State University, San Marcos, TX, USA.
出版信息
Methods Mol Biol. 2019;1855:363-370. doi: 10.1007/978-1-4939-8793-1_31.
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.
琼脂糖凝胶电泳技术的最新进展确定了使用更高电压和更短运行时间实现DNA和RNA高分辨率分离的条件。随后，我们开发了一种利用这些条件在琼脂糖凝胶上测量蛋白质与RNA配体结合亲和力的方法。这种天然凝胶迁移率变动分析方法非常容易操作，使用的是大多数实验室都有的常见分子生物学试剂。在这里，我们描述了进行天然琼脂糖凝胶电泳以表征蛋白质与RNA配体结合亲和力的方法。电泳时间少于10分钟，这最大限度地减少了蛋白质和配体的解离。我们使用p19 siRNA结合蛋白及其同源双链RNA配体，展示了使用这种琼脂糖凝胶迁移系统确定测量表观结合常数的最佳条件的策略。
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