Zheng Xuexiu, Cho Sunghee, Moon Heegyum, Loh Tiing Jen, Jang Ha Na, Shen Haihong
School of Life Science, Gwangju Institute of Science and Technology, 123 Cheomdan-gwagiro (Oryong-dong), Buk-gu, Gwangju, 500-712, South Korea.
Methods Mol Biol. 2016;1421:35-44. doi: 10.1007/978-1-4939-3591-8_4.
RNA-protein interaction can be detected by RNA pull-down and immunoblotting methods. Here, we describe a method to detect RNA-protein interaction using RNA pull down and to identify the proteins that are pulled-down by the RNA using immunoblotting. In this protocol, RNAs with specific sequences are biotinylated and immobilized onto Streptavidin beads, which are then used to pull down interacting proteins from cellular extracts. The presence of a specific protein is subsequently verified by SDS- polyacrylamide gel electrophoresis and immunoblotting with antibodies. Interactions between the SMN RNA and the PSF protein and between the caspase-2 RNA and the SRSF3 protein (SRp20) in nuclear extract prepared from HeLa cells are illustrated as examples.
RNA与蛋白质的相互作用可通过RNA下拉和免疫印迹法检测。在此，我们描述一种利用RNA下拉检测RNA与蛋白质相互作用，并通过免疫印迹鉴定被RNA下拉的蛋白质的方法。在本实验方案中，具有特定序列的RNA被生物素化并固定在链霉亲和素磁珠上，然后用于从细胞提取物中下拉相互作用的蛋白质。随后通过SDS-聚丙烯酰胺凝胶电泳和抗体免疫印迹来验证特定蛋白质的存在。以从HeLa细胞制备的核提取物中SMN RNA与PSF蛋白之间以及caspase-2 RNA与SRSF3蛋白（SRp20）之间的相互作用为例进行说明。
