磷酸化标签方法检测异染色质蛋白1αDNA损伤后蛋白磷酸化状态  

Phos-tag Based Analysis of Heterochromatin Protein-1α Phosphorylation after DNA Damage

作　　者：刘佳[1] 周光前[1,2] 尹献辉[2] 李雪芹[2] 王优雅[2] 胡静仪[2] 王子梅[2] 

机构地区：[1]四川大学华西医院生物治疗国家重点实验室,成都610041 [2]深圳大学医学院生物化学与分子生物学教研室

出　　处：《华西医学》2012年第6期828-831,共4页West China Medical Journal

基　　金：国家自然科学基金面上项目(81070270);深圳市科技基金基础研究计划面上项目(JC201005280552A)~~

摘　　要：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 introduce a Phos-tag based method for detecting phosphorylation state derived from heterochromatin protein-1α （HPlα following the DNA damage in mouse embryonic fibroblasts （MEFs）. Methods We carried out the isolation and primary culture of MEFs on the embryonic day 13.5 of C57 mice. Cells from two 100 mm dishes respectively for the control group and the test group were studied. Camptothecin （CPT） was used to perform DNA damage for cells in the test group, while cells in the control group were treated with the equal volume and concentration of dimethyl sulfoxide. Cellular proteins were separated by Phos-tag sodium dodecyl sulfate polyacrylamide gel electropheresis followed with immune-blotting. The membranes transferred by gel were incubated with the antibody against HP1α, and then with secondary antibodies conjugated with horseradish peroxidase. Phosphorylated and non-phosphorylated HPlα were detected and quantified by fluorescence scanning image system. Results A phosphorylated HP1α extra band was detected in the phos-tag western blotting, causing a mobility shift distinct from HP1α. Compared with the control group, Phosphorylated HP 1 α was transiently increased after DNA damage. Conclusions HP 1 α is phosphorylated following DNA damage, indicating that HPlct may play an important role in DNA damage repair process. On the other hand, application of Phos-tag based method is simple, convenient and practicable in determining protein＇s unknown phosphorylation, especially without the commercial phosphor-antibody.

关 键 词：蛋白质磷酸化 磷酸化标签 异染色质蛋白1α DNA损伤 

分 类 号：R341[医药卫生—基础医学]