番茄子叶总蛋白双向电泳体系的建立.pdf

园艺学报 2010 ,37 ( 4 ): 661 – 668
Acta Horticulturae Sinica
番茄子叶总蛋白双向电泳体系的建立
黎飞1,徐秋芳1,臧宪朋1,赖亿玉1,程维舜1,徐幼平2,*,蔡新忠1,*
(1浙江大学农业与生物技术学院生物技术研究所,杭州 310029;2浙江大学分析测试中心,杭州 310029)
摘要:通过对 IPG 胶条梯度、上样方式、上样量、聚焦条件等 4 方面条件的优化,建立了番茄子
叶总蛋白双向电泳体系,即采用 TCA 丙酮沉淀法提取番茄子叶总蛋白,选用 24 cm pH3-7NL 的 IPG 胶条,
采用水化上样,上样量 300 μg,按聚焦程序Ⅰ或Ⅱ聚焦后进行双向电泳分离和银染染色。若采用制备胶,
以获取高含量蛋白点,则将上样量提高至 mg,按聚焦程序Ⅲ进行聚焦后进行双向分离,并采用胶体
考马斯亮蓝染色。采用该优化的体系可以获得分辨率高、重复性好的双向电泳图谱。
关键词:番茄;双向电泳;条件优化;子叶;蛋白质
中图分类号:S 文献标识码:A 文章编号:0513-353X(2010)04-0661-08

Establishment of Two-dimensional Electrophoresis System of Tomato
Cotyledons
LI Fei1,XU Qiu-fang1,ZANG Xian-peng1,LAI Yi-yu1,CHENG Wei-shun1,XU You-ping2,*,and CAI
Xin-zhong1,*
(1Institute of Biotechnology,College of Agriculture and Biotechnology,Zhejiang University,Hangzhou 310029,China;
2Centre of Analysis and Measurement,Zhejiang University,Hangzhou 310029,China)
Abstract:A two-dimensional electrophoresis(2-DE)system for proteomic analysis of tomato
cotyledons was established by optimizing the parameters including the pH gradient of IPG strip,sample
application,sample loading and isoelectric focusing conditions. The optimized system include the
following steps:Extracting the total protein from tomato cotyledons by TCA/acetone precipitation,
separating the proteins with 24 cm pH3-7NL IPG strips,loading protein samples of 300 μg by reh