双向凝胶电泳比较三种常用蛋白质提取方法

ISS N　100727626 C N　1123870ΠQ

中国生物化学与分子生物学报

Chinese Journal of Biochemistry and M olecular Biology

2005年10月

21(5):691～694

・技术与方法・

双向凝胶电泳比较三种常用蛋白质提取方法

翁　瑜1),2),　曾群力2),3),　姜　槐2),　许正平2),3)3

(1)浙江大学生命科学学院;2)浙江大学医学院浙江省生物电磁学重点研究实验室;3)浙江大学医学院环境基因组学研究中心,杭州　310031)

摘要　组织(或细胞)的蛋白质提取效率直接影响蛋白质双向凝胶电泳(22DE)的分辨率.为探索建立适用于人乳腺癌细胞株MCF27蛋白质提取的最佳条件,比较目前在双向凝胶电泳中常用的3种蛋白质提取方法对MCF27细胞总蛋白的提取效率.MCF27细胞经培养后,分别采用M2PER试剂、标准裂解液或含硫脲裂解液提取其总蛋白质,然后进行双向凝胶电泳,并根据凝胶上蛋白质斑点的丰度和分布特点判断所得双向电泳图谱的质量,以确定MCF27细胞蛋白质提取的相对最佳方法.结果显示,M2PER试剂法得到的图谱分辨率较低,蛋白质主要集中分布在分子量15～70kD,pH417～613的范围内;标准裂解液法得到的图谱分辨率有所提高,蛋白质分布比M2PER试剂法得到的图谱广;硫脲裂解液法得到的图谱是三者中分辨率最高的,尤其是高丰度蛋白和高分子量蛋白分离效果比前两者好.结果表明,在3种常用的蛋白质提取方法中,硫脲裂解液对细胞蛋白质的溶解性最佳,相对更适合于提取MCF27细胞的蛋白质,并与双向凝胶电泳条件更兼容.

关键词　蛋白质提取,双向凝胶电泳,MCF27,条件优化

中图分类号　Q503

Comparison of Three Protein Extraction Methods by Tw o2

Dimensional E lectrophoresis

WE NG Y u1),2),ZE NG Qun2Li2),3),J I ANG Huai2),X U Zheng2Ping2),3)3

(1)College o f Life Sciences,2)Bioelectromagnetics Laboratory,3)Research Center for Environmental G enomics,

Zhejiang Univer sity School o f Medicine,Hangzhou　310031,China)

Abstract　Protein extraction from tissue or cells is a key step to achieve high2res olution protein separation in tw o dimensional electrophoresis(22DE).Three routine cellular total protein extraction methods were com pared in order to determine an optimal one for human breast cancer cell line MCF27.The cultured MCF27cells were lysed by M2PER kit,standard lysis buffer or im proved lysis buffer,respectively.Then the extracted total proteins were subjected to22DE,and the best extraction method was determined by the indexes of protein distribution and abundance on corresponding silver2stained gel.Data showed that use of M2PER kit gave the lowest res olution,in which m ost proteins were distributed in the pI ranging from417to613with m olecular weight between15kD and70kD.Standard lysis bu ffer im proved protein res olution with broader protein distribution pattern.Im proved lysis bu ffer generated the best res olution am ong these three methods,especially for the high2abundance and high m olecular weight proteins.Based on above results,we concluded that the im proved lysis bu ffer has the best protein s olubilization ability,which renders it much m ore suitable for cellular protein extraction from MCF27,and is m ore com patible with the conditions of22DE.

K ey w ords　protein extraction,tw o dimensional electrophoresis,MCF27,optimization

收稿日期:2004212203,接收日期:2005203221

国家自然科学基金项目(N o.50137030,30170792),浙江省自然科学基金项目(N o.301524)和浙江省卫生厅重点项目(N o.2004Z D006)资助

3联系人　T el:0571287217386,Fax:0571287217410,E2mail:zpxu@

Received:December3,2004;Accepted:M arch21,2005

Supported by National Natural Science F oundation of China(N o.50137030,30170792),and Natural Science F oundation of Zhejiang Province(N o.301524),and K ey Program of Health Bureau of Zhejiang Province(N o.2004Z D006)

3C orresponding author T el:0571287217386,Fax:0571287217410,E2mail:zpxu@