适于双向电泳分析的番茄叶片总蛋白提取方法的优化  

Optimization of total protein extraction from tomato leaves for two-D gel electrophoresis analysis

作　　者：徐幼平[1] 徐秋芳[2] 蔡新忠[2] 

机构地区：[1]浙江大学分析测试中心,浙江杭州310029 [2]浙江大学生物技术研究所植物保护系,浙江杭州310029

出　　处：《浙江农业学报》2007年第2期71-74,共4页Acta Agriculturae Zhejiangensis

基　　金：国家自然科学基金资助项目(30200179)

摘　　要：比较了三氯乙酸(TCA)、酚和十二烷基硫酸钠(SDS)3种提取方法在所获番茄叶片总蛋白产量及其在SDS聚丙烯酰胺凝胶电泳分辨率等方面的区别,并对3种蛋白溶解液以及超声处理在蛋白质溶解中的作用进行了分析探讨。结果表明,TCA提取法最佳,该方法提取番茄叶片总蛋白的得率最高、所获蛋白在SDS聚丙烯酰胺凝胶电泳中形成条带数目最多,最清晰;酚法次之;SDS法最差。由离液剂和去污剂组成的蛋白溶解液复溶蛋白沉淀所获蛋白产量显著高于Tris-NaCl溶解液。还原剂和两性电解质以及超声处理显著增进蛋白质的溶解,提高蛋白产量。采用TCA提取法获得蛋白沉淀,再以溶解液Ⅱ(7 mol/L尿素,2 mol/L硫尿,2%CHAPS,2%SB3-10,65 mmol/L DTT,0.2%两性电解质)溶解,结合超声助溶的方法体系可以获得高得率和高电泳分辨率的番茄叶片总蛋白样品,该方法适用于双向电泳分析等下游操作。In this study, three protein extraction methods based on trichloroacetic acid （TCA）, phenol and sodium dodecyl sulfate （SDS） respectively, were compared in total protein yield from tomato leaf and the protein resolution in SDS-polyacrylamide gel electrophoresis （PAGE）. Effect of three solubilization buffers and supersonic treatment on protein solubilization was analyzed. It was fotmd that the TCA method was the best one resulting in the highest total protein yield, the most protein bands and the best resolution in SDS-PAGE, the phenol method was less good, while the SDS method was the worst one. The solubilization buffer consisting of chaotropes and detergents resolubilized protein pellet much better and thus resulting in much higher protein yield compared with the one consisting of Tris-NaCl. Addition of a reducing agent and an ampholyte, and supersonic treatment significantly enhanced protein solubilization. Taken together, a tomato leaf total protein isolation approach, resulting in high protein yield and high resolution in SDS-PAGE, and suitable for the downstream manipulation such as two-D gel electrophoresis analysis, was established, which consists of the protein extraction by the TCA- based method, followed by re-solubilization of the protein pellets with the solubilization buffer consisting of 7 mol/L urea, 2 mol/L thiourea, 2 % CHAPS, 2 % SB3-10,65 mmol/L DTY,0. 2 % ampholyte （pH 3 - 10）, in combination with supersonic treatment.

关 键 词：番茄 蛋白 双向电泳 三氯乙酸 两性电解质 超声处理 

分 类 号：Q503[生物学—生物化学]