Yakhnin Alexander V, Yakhnin Helen, Babitzke Paul
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, USA.
Methods Mol Biol. 2012;905:201-11. doi: 10.1007/978-1-61779-949-5_12.
The gel mobility shift assay is a powerful technique for detecting and quantifying protein-RNA interactions. While other techniques such as filter binding and isothermal titration calorimetry (ITC) are available for quantifying protein-RNA interactions, gel shift analysis provides the added advantage that you can visualize the protein-RNA complexes. In the gel shift assay, protein-RNA complexes are typically separated from the unbound RNA using native polyacrylamide gels in Tris/borate/EDTA buffer, although an alternative Tris-glycine buffering system is superior in many situations. Here, we describe both gel shift methods, along with strategies to improve separation of protein-RNA complexes from free RNA, which can be a particular challenge for small RNA binding proteins.
凝胶迁移率变动分析是一种用于检测和定量蛋白质-RNA相互作用的强大技术。虽然还有其他技术如滤膜结合法和等温滴定量热法（ITC）可用于定量蛋白质-RNA相互作用，但凝胶迁移分析具有额外的优势，即可以可视化蛋白质-RNA复合物。在凝胶迁移分析中，通常使用Tris/硼酸/EDTA缓冲液中的天然聚丙烯酰胺凝胶将蛋白质-RNA复合物与未结合的RNA分离，不过在许多情况下，另一种Tris-甘氨酸缓冲系统更为优越。在此，我们描述了这两种凝胶迁移方法，以及从游离RNA中改善蛋白质-RNA复合物分离的策略，这对于小RNA结合蛋白可能是一个特别的挑战。
