Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA; Department of Neuroscience, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA.
Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA; Department of Neuroscience, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA.
STAR Protoc. 2024 Jun 21;5(2):103013. doi: 10.1016/j.xpro.2024.103013. Epub 2024 Apr 11.
DNA-binding proteins perform diverse functions, including regulating cellular growth and orchestrating chromatin architecture. Here, we present a protocol to discover proteins specifically interacting with a hexanucleotide repeat DNA, the expansion of which is known as the most frequent genetic cause of familial C9orf72 amyotrophic lateral sclerosis and frontotemporal dementia. We describe steps to fish out DNA-binding proteins recognizing double-stranded repeat DNAs using a SILAC (stable isotope labelling by amino acids in cell culture)-based approach and validate the results using electrophoretic mobility shift assay. For complete details on the use and execution of this protocol, please refer to Liu et al..
DNA 结合蛋白具有多种功能，包括调节细胞生长和调控染色质结构。在这里，我们提供了一种发现与六核苷酸重复 DNA 特异性相互作用的蛋白质的方案，该重复 DNA 的扩增是家族性 C9orf72 肌萎缩侧索硬化症和额颞叶痴呆最常见的遗传原因。我们描述了使用基于 SILAC（稳定同位素标记的细胞培养中的氨基酸）的方法钓取识别双链重复 DNA 的 DNA 结合蛋白的步骤，并使用电泳迁移率变动分析验证结果。有关该方案使用和执行的完整详细信息，请参阅 Liu 等人的文章。
