Ozolinš Terence R S
Department of Pharmacology and Toxicology, Queen's University, Kingston, ON, Canada.
Methods Mol Biol. 2012;889:291-303. doi: 10.1007/978-1-61779-867-2_18.
The electrophoretic mobility shift assay (EMSA) is a sensitive relatively straightforward methodology used to detect sequence-specific DNA-protein interactions. It is the fundamental procedure of several variants that allow qualitative and quantitative assessments of protein-nucleic acid complexes. Classically, nuclear proteins and DNA are combined and the resulting mixture is electrophoretically separated in polyacrylamide or agarose gel under native conditions. The distribution within the gel is generally detected with autoradiography of the ³²P-labeled DNA. The underlying principle is that nucleic acid with protein bound to it will migrate more slowly through a gel matrix than the free nucleic acid. In this chapter, a representative protocol is described that addresses specific challenges of using whole embryos as the nuclear protein source, and the most common and informative EMSA variant, the "supershift," is also presented. The important points are underscored and approaches for troubleshooting are explained. References are provided for alternative methods and extensions of the basic protocol.
电泳迁移率变动分析（EMSA）是一种灵敏且相对简单的方法，用于检测序列特异性DNA-蛋白质相互作用。它是几种变体的基本程序，可对蛋白质-核酸复合物进行定性和定量评估。传统上，将核蛋白与DNA混合，然后在天然条件下于聚丙烯酰胺或琼脂糖凝胶中对所得混合物进行电泳分离。凝胶内的分布通常通过对³²P标记的DNA进行放射自显影来检测。其基本原理是，与蛋白质结合的核酸在凝胶基质中的迁移速度比游离核酸慢。在本章中，描述了一个代表性方案，该方案解决了使用全胚胎作为核蛋白来源的特定挑战，还介绍了最常见且信息丰富的EMSA变体“超迁移”。强调了要点并解释了故障排除方法。提供了替代方法和基本方案扩展的参考文献。
