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目的通过检测异染色质蛋白1α(HP1α)在DNA损伤后的磷酸化状况,介绍一种用磷酸化标签(phos-tag)试剂检测磷酸化蛋白质的新方法。方法取雄雌C57小鼠交配后孕13.5 d胚胎,分离并原代培养小鼠胚胎成纤维细胞。对照组及实验组(6个损伤时间点)各取2个100 mm培养皿的细胞进行实验,实验组细胞用喜树碱进行DNA损伤;对照组用等量的二甲基亚砜处理。用掺入phos-tag的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离蛋白并转印,将膜用抗HP1α的抗体孵育,用偶联辣根过氧化物酶的抗体做二抗,通过成像系统检测蛋白。结果实验组存在一条与HP1α有明显不同迁移率的磷酸化HP1α条带,与对照组相比DNA损伤后磷酸化HP1α含量一过性增多。结论 HP1α被DNA损伤诱导为磷酸化状态,提示其可能在DNA修复过程中扮演重要角色。Phos-tag蛋白质印迹法可采用普通抗体检测磷酸化的蛋白,是一种简便易行的检测未知磷酸化蛋白质的新方法。
OBJECTIVE: To detect the phosphorylation status of heterochromatin 1α (HP1α) after DNA damage, a new method for detecting phosphorylated protein by using phos-tag reagent was introduced. METHODS: Female and male C57 mice were mated to 13.5 days post embryo embryos, and primary cultured mouse embryonic fibroblasts were isolated. The control group and the experimental group (6 injury time points) each took two 100 mm culture dish cells for experiments. The cells in the experimental group were subjected to DNA damage with camptothecin. The control group was treated with the same amount of dimethyl sulfoxide. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis incorporating phos-tag and transferred, the membrane was incubated with anti-HPl [alpha] antibody, secondary antibody with horseradish peroxidase-conjugated antibody, Protein detection by imaging system. Results There was a phosphorylated HP1α band in the experimental group that had a significantly different mobility from HP1α. Compared with the control group, the content of phosphorylated HP1α in the experimental group increased transiently. Conclusion HP1α is induced to phosphorylation by DNA damage, suggesting that HP1α may play an important role in DNA repair. Phos-tag Western blotting can be used to detect phosphorylated protein by ordinary antibody, which is a new and easy way to detect unknown phosphorylated protein.