大鼠脑内突触后致密物蛋白质的提取及鉴定  

Extraction and identification of postsynaptic density protein from rat brain

作　　者：周颖[1] 黄志凌[1] 肖波[2] 吴小妹[2] 吴刘亦文[1] 杨璞[1] 

机构地区：[1]中南大学湘雅二医院神经内科,湖南长沙410011 [2]中南大学湘雅医院神经内科,湖南长沙410008

出　　处：《基础医学与临床》2009年第3期305-308,共4页Basic and Clinical Medicine

基　　金：国家自然科学基金青年基金(30500172)

摘　　要：目的提取纯度高、易分离溶解的突触后致密物(PSD)蛋白质。方法应用蔗糖密度梯度离心法,逐次获取P2,突触小体和PSD-1;应用Western blot检测PSD-1蛋白质的纯度;对PSD-1运用膜顺序提取法获取可溶性蛋白PSD-2;应用双向凝胶电泳技术分离PSD-1与PSD-2蛋白质点,PDQuest软件分析比较其数目差异。结果突触后标志分子PSD-95在P2成分、突触体和PSD-1 3组成分中均呈免疫染色阳性,而突触前标志分子突触素在PSD-1成分中呈免疫阴性。从PSD-1蛋白中电泳分离到286±25个蛋白质点而显著低于从PSD-2中分离到720±17个蛋白质点(P<0.01)。结论蔗糖密度梯度离心联合膜顺序提取法可获得纯度高、易分离溶解的PSD蛋白质。Objective To purify postsynaptic density （PSD） proteins with better dissolubility. Methods The constituents of P2, synaptosome and PSD-1 were obtained by sucrose gradient centrifugation. The purity of PSD-1 protein was validated by Western blot. These soluble proteins PSD-2 were extracted from PSD-1 using the technology of membrane sequence extraction. The protein spots of PSD-1 or PSD-2 were seperated by the two-dimensional gel electrophoresis （2-DE） and analyzed by＇ the software of PDQuest to show the different dissolubility of PSD-1 or PSD- 2. Results The results of Western blot showed that the postsynaptic marker PSD-95 were positive staining in P2, synaptosome and PSD-1, but the presynaptic marker synaptophysin was not seen in PSD-1. There were 286 ± 25 protein spots in the electrophoregram of PSD-1, 720 ± 17 protein spots in the electrophoregram of PSD-2, and the difference was significant （ P 〈 0. 01 ）. Conclusion Pure PSD proteins can be obtained and dissolved easily by the method of sucrose gradient centrifugation plus membrane sequence extraction.

关 键 词：突触后致密物 蔗糖密度梯度离心 膜顺序提取 双向凝胶电泳 

分 类 号：R363[医药卫生—病理学]