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

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.