HeLa细胞蛋白质组双向电泳技术的建立

目的:建立和优化HeLa细胞蛋白质组分析所需的样品处理方法和双向电泳技术.方法:用三种不同配方的裂解液提取HeLa细胞中的蛋白质,进行双向凝胶电泳,用Amersham ImageMaster VDS-CL型凝胶成像系统获取凝胶图像,以PDQuest(7.4.0)软件进行比较分析.结果:联合使用硫脲和尿素有利于蛋白质的提取,增加碱性蛋白点的检出数量;广谱蛋白酶抑制剂的使用可大大增加双向电泳可检出的蛋白点数,同时使可检出的碱性蛋白点明显增加.结论:本文建立了HeLa细胞双向电泳分析方法,提高了2-DE图谱中蛋白位点的分辨率和重复性,获得了较为理想、清晰的双向电泳图谱,为其蛋白质组学进一步研究奠定了基础.

Establishment of a two-dimensional electrophoresis technique for proteome of HeLa cells

Objective:To establish and optimize the two-dimensional gel electrophoresis(2-DE)and the preparation of protein samples for the proteome of HeLa cells.Methods:The proteins of HeLa cell were extracted by three different lysis buffers to do 2-DE.The gel images then were acquired by Amersham ImageMaster VDS-CL to do the comparative analysis with PDQuest(7.4.0)software.Results:The combination of Thiourea and Urea can be favorable for the extraction of proteins,and increase the amount of the detectable spots for the alkaline protein.The application of broad-spectrum protease inhibitor could increase the amount of the detectable protein spots in 2-DE remarkably,and also the alkaline protein spots.Conclusions:A two-dimensional electrophoresis technique for HeLa cells was established and an ideal 2-DE map with higher resolution and better reproducibility for HeLa cells was obtained clearly,which laid a foundation for the further studies on proteome of HeLa cells.