Key Laboratory of Molecular Biology and Gene Engineering in Jiangxi Province, College of Life Science, Nanchang University, Jiangxi 330031, China.
Key Laboratory of Molecular Biology and Gene Engineering in Jiangxi Province, College of Life Science, Nanchang University, Jiangxi 330031, China.
STAR Protoc. 2024 Mar 15;5(1):102818. doi: 10.1016/j.xpro.2023.102818. Epub 2024 Jan 5.
Long non-coding RNAs (lncRNAs) work together with diverse RNA-binding proteins (RBPs) to fulfill key regulations in important cellular functions. Here, we present a protocol to detect lncRNA-RBP interactions in vitro using a tRNA scaffold containing a streptavidin aptamer pull-down assay. We describe steps for preparing both protein and lncRNA transcripts, lncRNA-protein interaction detection with an in vitro binding assay, and western blot analysis. This protocol is applicable to screen for RNA-interacting proteins using cell lysates followed by mass spectrometry analysis. For complete details on the use and execution of this protocol, please refer to Yang et al. (2023)..
长非编码 RNA(lncRNA)与多种 RNA 结合蛋白(RBP)共同作用,以实现重要细胞功能中的关键调控。在此,我们提供了一种使用含链霉亲和素适体的 tRNA 支架在体外检测 lncRNA-RBP 相互作用的方案。我们描述了制备蛋白质和 lncRNA 转录本的步骤、通过体外结合测定进行 lncRNA-蛋白质相互作用检测以及 Western blot 分析。该方案适用于使用细胞裂解物进行筛选,然后进行质谱分析以筛选与 RNA 相互作用的蛋白质。有关此方案的使用和执行的完整详细信息,请参阅 Yang 等人(2023 年)。