Hou Shuai, Shi Lei, Lei Haixin
Institute of Cancer Stem Cell, Cancer Center, Dalian Medical University, No. 9 West Section Lvshun South Road, Dalian, Liaoning, 116044, People's Republic of China.
Methods Mol Biol. 2016;1421:23-34. doi: 10.1007/978-1-4939-3591-8_3.
RNA-protein complexes are essential for the function of different RNAs, yet purification of specific RNA-protein complexes can be complicated and is a major obstacle in understanding the mechanism of regulatory RNAs. Here we present a protocol to purify RNA-protein complexes assembled in vitro based on biotin-streptavidin affinity. In vitro transcribed RNA is labeled with (32)P and biotin, ribonucleoprotein particles or RNPs are assembled by incubation of RNA in nuclear extract and fractionated using gel filtration, and RNP fractions are pooled for biotin-streptavidin affinity purification. The amount of RNA-protein complexes purified following this protocol is sufficient for mass spectrometry.
RNA-蛋白质复合物对于不同RNA的功能至关重要，然而，特定RNA-蛋白质复合物的纯化可能很复杂，并且是理解调控RNA机制的主要障碍。在此，我们提出一种基于生物素-链霉亲和素亲和力来纯化体外组装的RNA-蛋白质复合物的方法。体外转录的RNA用(32)P和生物素标记，通过将RNA在核提取物中孵育来组装核糖核蛋白颗粒或RNP，并使用凝胶过滤进行分级分离，然后将RNP级分合并用于生物素-链霉亲和素亲和纯化。按照此方法纯化的RNA-蛋白质复合物的量足以用于质谱分析。
