Institute of Medical Sciences, School of Medicine, Medical Sciences & Nutrition, University of Aberdeen, Aberdeen, Scotland, UK.
Methods Mol Biol. 2023;2603:209-218. doi: 10.1007/978-1-0716-2863-8_17.
DNA replication is a highly complex process that achieves the faithful transmission of genetic information from parent to progeny. Recruitment of DNA replication proteins to DNA is dynamically regulated during the cell cycle and in response to replication stresses. For a large-scale analysis of DNA replication proteins, I established a method for analysis of chromatin-bound proteins by SILAC (stable isotope labeling by amino acids in cell culture)-based quantitative proteomics. Here I describe a detailed methodology for SILAC labeling of budding yeast Saccharomyces cerevisiae, then nuclear isolation and chromatin preparation from synchronized yeast cells, prior to quantitative proteomic analysis of DNA replication proteins.
DNA 复制是一个高度复杂的过程，它实现了遗传信息从亲代到后代的忠实传递。在细胞周期中以及对复制应激的反应中，DNA 复制蛋白被动态调节招募到 DNA 上。为了对 DNA 复制蛋白进行大规模分析，我建立了一种基于 SILAC（稳定同位素标记的氨基酸细胞培养）的定量蛋白质组学方法，用于分析染色质结合蛋白。在这里，我描述了一种详细的方法，用于对酿酒酵母 Saccharomyces cerevisiae 进行 SILAC 标记，然后对同步化酵母细胞进行核分离和染色质制备，最后对 DNA 复制蛋白进行定量蛋白质组学分析。
