Electrophoretic mobility shift assays for RNA-protein complexes.
Electrophoretic mobility shift assays for RNA-protein complexes.
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DOI：
10.1101/pdb.prot080721
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发表时间：
2014-04-01
影响因子：
--
通讯作者：
Rio, Donald C
中科院分区：
文献类型：
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作者：
Rio, Donald C
电泳迁移迁移测定（EMSA），或凝胶迁移迁移测定，是一种流行的和强大的技术，用于检测rna -蛋白质相互作用。它依赖于这样一个事实：裸RNA在非变性凝胶上具有一定的流动性，但如果RNA与蛋白质结合，RNA的流动性就会降低。因此，蛋白质的结合导致凝胶上RNA的特征向上移动，使用放射性标记的RNA进行监测。由于尚未完全了解的原因，大多数rna -蛋白复合物-特别是那些由高亲和力相互作用产生的复合物-在电泳分离所需的较长时间内不会解离。由于高亲和相互作用更稳定，因此通常可以在弱相互作用的“背景”上识别特定的相互作用。因此，emsa可以使用复杂的蛋白质混合物（如细胞提取物）进行。它们可用于研究广泛的rna -蛋白质相互作用——从单个蛋白质结合事件到大型复合物（如剪接体）的组装。emsa也可用于确定rna -蛋白质相互作用的动力学参数（如亲和常数）。
The electrophoretic mobility shift assay (EMSA), or gel mobility shift assay, is a popular and powerful technique for the detection of RNA-protein interactions. It relies on the fact that naked RNA has certain mobility on nondenaturing gels, but if the RNA is bound by protein, the mobility of the RNA is reduced. Therefore, the binding of protein results in a characteristic upward shift of the RNA on a gel, as monitored using radiolabeled RNA. For reasons that are not completely understood, most RNA-protein complexes--particularly those that result from high-affinity interactions--do not dissociate during the prolonged times required for electrophoretic separation. Because high-affinity interactions are more stable, it is often possible to identify specific interactions over a "background" of weak interactions. Accordingly, EMSAs can be performed using complex mixtures of proteins such as cell extracts. They can be used to investigate a wide range of RNA-protein interactions--from single protein-binding events to assembly of large complexes such as the spliceosome. EMSAs can also be useful for determining kinetic parameters (such as affinity constants) for RNA-protein interactions.