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ISSN　100727626CN　1123870ΠQ

中国生物化学与分子生物学报

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,　许正平

2)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,度和分布特点判断所得双向电泳图谱的质量,以确定27.结果显示,M2PER试剂法得到的图谱分辨率较低,～kD,pH417～613的范围内;M2PER试剂法得到的图谱广;比前两者好.,,硫脲裂解液对细胞蛋白质的溶解性最佳,27,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

ComparisonofThreeProteinExtractionMethodsbyTwo2

DimensionalElectrophoresis

WENGYu

1),2)

,ZENGQun2Li

2)

2),3)

,JIANGHuai,XUZheng2Ping

)

2)2),3)3

(1)CollegeofLifeSciences,BioelectromagneticsLaboratory,3ResearchCenterforEnvironmentalGenomics,

ZhejiangUniversitySchoolofMedicine,Hangzhou　310031,China)

Abstract　Proteinextractionfromtissueorcellsisakeysteptoachievehigh2resolutionproteinseparationin

twodimensionalelectrophoresis(22DE).ThreeroutinecellulartotalproteinextractionmethodswerecomparedinordertodetermineanoptimaloneforhumanbreastcancercelllineMCF27.TheculturedMCF27cellswerelysedbyM2PERkit,standardlysisbufferorimprovedlysisbuffer,respectively.Thentheextractedtotalproteinsweresubjectedto22DE,andthebestextractionmethodwasdeterminedbytheindexesofproteindistributionandabundanceoncorrespondingsilver2stainedgel.DatashowedthatuseofM2PERkitgavethelowestresolution,inwhichmostproteinsweredistributedinthepIrangingfrom417to613withmolecularweightbetween15kDand70kD.Standardlysisbufferimprovedproteinresolutionwithbroaderproteindistributionpattern.Improvedlysisbuffergeneratedthebestresolutionamongthesethreemethods,especiallyforthehigh2abundanceandhighmolecularweightproteins.Basedonaboveresults,weconcludedthattheimprovedlysisbufferhasthebestproteinsolubilizationability,whichrendersitmuchmoresuitableforcellularproteinextractionfromMCF27,andismorecompatiblewiththeconditionsof22DE.Keywords　proteinextraction,twodimensionalelectrophoresis,MCF27,optimization

收稿日期:2004212203,接收日期:2005203221

国家自然科学基金项目(No.50137030,30170792),浙江省自然科学基金项目(No.301524)和浙江省卫生厅重点项目(No.2004ZD006)资助

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联系人　Tel:0571287217386,Fax:0571287217410,E2mail:zpxu@zju.edu.cn

Received:December3,2004;Accepted:March21,2005

SupportedbyNationalNaturalScienceFoundationofChina(No.50137030,30170792),andNaturalScienceFoundationofZhejiangProvince(No.301524),andKeyProgramofHealthBureauofZhejiangProvince(No.2004ZD006)

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CorrespondingauthorTel:0571287217386,Fax:0571287217410,E2mail:zpxu@zju.edu.cn