大豆花瓣蛋白双向电泳分析技术体系的优化  

Optimization of Two-Dimensional Gel Electrophoresis for Proteome of Soybean(Glycine Max L.Merril) Floret

作　　者：郑天慧[1] 宋波[1] 姜自芹[1] 刁桂珠[1] 曾蕊[1] 吴帅[1] 拓云[1] 刘珊珊[1] 

机构地区：[1]东北农业大学

出　　处：《哈尔滨师范大学自然科学学报》2010年第6期75-80,共6页Natural Science Journal of Harbin Normal University

基　　金：国家自然科学基金资助项目(31071440);黑龙江省普通高等学校青年骨干支持计划项目(1155G12);博士后研究人员落户黑龙江科研启动项目(2009HB009);转基因生物新品种培育重大专项子任务:抗病虫转基因大豆新品种培育资助项目(2008ZX08004-004)

摘　　要：双向电泳技术体系的优化是蛋白质组学研究的前提和基础.本文针对大豆花瓣含有大量色素和酚等干扰物质的现象,比较6种蛋白提取方法和2种蛋白裂解液配方,优化胶条范围和等电聚焦程序.结果表明:pH为4～7 IPG胶条适于大豆全花蛋白分离;TCA/丙酮+2D Clean Up试剂盒法去除杂质充分,提取的蛋白含量高、纯度好、分离效果佳、耗时短;蛋白裂解液采用5 mol/L尿素,2 M硫脲,2%CHAPS,2%SB3-10,5 mmol/L TCEP,20 mmol/L DTT,0.5%IPG Buffer为好;增加离心转速、时间和丙酮清洗次数能促进蛋白溶解,增加蛋白纯度,初步建立了一套适合大豆全花蛋白双向电泳分析的技术体系.Protein extraction methods and electrophoresis conditions are the key of two dimensional electrophoresis technologies in proteomics research.A two dimensional electrophoresis technology system for soybean full flower protein is established.There are many pigments,phenol and other interfering substances in the soybean petals,so six protein extraction methods and two protein analysis buffer are compared to improve protein extraction methods,and the range of IPG strip and isoelectric focusing procedure are optimized.The results show that TCA / acetone ＋2 D Clean Up kit remove most impurities,yield the high protein content,good separation and short time-consuming;The analysis buffer containing 5 mol/L Urea,2 mol/L Thiourea,2% CHAPS,2% SB3-10,5 mmol/L TCEP, 20 mmol/L DTT,0.5% IPG Buffer has good dissolving ability.Increasing the centrifugation speed,time,and the times of acetone washing can promote the dissolving of protein and increase protein purity.pH 4~7 IPG strip is suitable for separation of soybean full flower protein.A two dimensional electrophoresis proteome analysis technology system for soybean full flower proteins is established.

关 键 词：大豆花瓣 双向电泳 技术体系 优化 

分 类 号：Q510.3[生物学—生物化学]