Yeh Hsin-Sung, Chang Jae-Woong, Yong Jeongsik
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota Twin Cities, 6-155 Jackson Hall, 321 Church Street SE, Minneapolis, MN, 55455, USA.
Methods Mol Biol. 2016;1421:165-74. doi: 10.1007/978-1-4939-3591-8_14.
Characterizing protein-protein and protein-RNA interaction networks is a fundamental step to understanding the function of an RNA-binding protein. In many cases, these interactions are transient and highly dynamic. Therefore, capturing stable as well as transient interactions in living cells for the identification of protein-binding partners and the mapping of RNA-binding sequences is key to a successful establishment of the molecular interaction network. In this chapter, we will describe a method for capturing the molecular interactions in living cells using formaldehyde as a crosslinker and enriching a specific RNA-protein complex from cell extracts followed by mass spectrometry and Next-Gen sequencing analyses.
表征蛋白质-蛋白质和蛋白质-RNA相互作用网络是理解RNA结合蛋白功能的基本步骤。在许多情况下，这些相互作用是短暂且高度动态的。因此，在活细胞中捕获稳定和短暂的相互作用以鉴定蛋白质结合伴侣并绘制RNA结合序列图谱，是成功建立分子相互作用网络的关键。在本章中，我们将描述一种使用甲醛作为交联剂在活细胞中捕获分子相互作用，并从细胞提取物中富集特定RNA-蛋白质复合物，随后进行质谱分析和新一代测序分析的方法。
