使用双滤膜结合测定法，随后进行磷光成像或高通量测序来测量蛋白质-RNA结合的实验方案。
Protocol to measure protein-RNA binding using double filter-binding assays followed by phosphorimaging or high-throughput sequencing.
作者信息
Vega-Badillo Joel, Zamore Phillip D, Jouravleva Karina
机构信息
RNA Therapeutics Institute, University of Massachusetts Chan Medical School, 368 Plantation Street, Worcester, MA 01605, USA.
RNA Therapeutics Institute, University of Massachusetts Chan Medical School, 368 Plantation Street, Worcester, MA 01605, USA; Howard Hughes Medical Institute, University of Massachusetts Chan Medical School, 368 Plantation Street, Worcester, MA 01605, USA.
出版信息
STAR Protoc. 2023 Jun 3;4(2):102336. doi: 10.1016/j.xpro.2023.102336.
Binding affinity quantitatively describes the strength of a molecular interaction and is reported by the equilibrium dissociation constant (K). Here, we present a protocol to measure K of mammalian microRNA-loaded Argonaute2 protein by double filter binding. We describe steps for radiolabeling target RNA, measuring concentration of binding-competent protein, setting up binding reactions, separating protein-bound RNA from protein-unbound RNA, preparing library for Illumina sequencing, and performing data analysis. Our protocol is easily applied to other RNA- or DNA-binding proteins. For complete details on the use and execution of this protocol, please refer to Jouravleva et al..
结合亲和力定量描述分子相互作用的强度，并通过平衡解离常数（K）来表示。在此，我们介绍一种通过双重过滤结合来测量负载哺乳动物微小RNA的AGO2蛋白的K值的方法。我们描述了对靶RNA进行放射性标记、测量具有结合能力的蛋白质浓度、设置结合反应、将与蛋白质结合的RNA与未结合蛋白质的RNA分离、制备用于Illumina测序的文库以及进行数据分析的步骤。我们的方法可轻松应用于其他RNA或DNA结合蛋白。有关本方法的使用和执行的完整详细信息，请参考Jouravleva等人的文献。
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