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作 者 ：于冯,郑春芳,施孟如,刘伟成,仇建标,冀德伟,黄丽,陈少波

期 刊 ：热带亚热带植物学报 2011年 19卷 6期 页码：519~523

关键词：秋茄；蛋白质；提取；双向电泳(2-DE)；技术优化；

Keywords:Kandelia candel, Protein, Extraction, Two-dimensional electrophoresis (2-DE), Optimization,

摘 要 ：对适用于秋茄(Kandelia candel)叶片蛋白质组学研究的双向电泳技术体系进行了优化。结果表明,采用酚抽提法提取的蛋白质浓度较低,约1.7 μg μL^-1,SDS-PAGE电泳后几乎无条带;而采用改良TCA-丙酮沉淀法可显著提高提取液中蛋白质的含量,可达12.4 μg μL^-1。秋茄叶片蛋白质主要分布在pH 4~7的范围内,最适于采用pH 3~10 NL的胶条,17 cm胶条的最适上样量为400 μg,经银染后可得蛋白点约(564±34)个。用TCA-丙酮沉淀法提取秋茄叶片全蛋白质组,按优化的参数进行双向电泳分离,获得了高分辨率、高重复性的图谱,可用于秋茄蛋白质组学研究。

Abstract:In order to study the proteome of Kandelia candel leaves, a two-dimensional electrophoresis (2-DE) system was established and optimized. Compared with the low protein content (1.7 μg μL^-1) extracted by phenol (Phe), the improved trichloroacetic acid/acetone precipitation (TCA) method could obviously increase the extracting efficiency up to 12.4 μg μL^-1. The protein spots of Kandelia candel leaves mainly distributed in the range of pH 4-7. The optimal loading quantity of samples were 400 μg gel^-1 for a 17 cm IPG strip (pH 3-10 NL), which can detect 564±34 protein spots after silver staining. Therefore, the high resolution and repeatable 2-DE map could be obtained by using TCA extract method and the optimum system, which could widely use in proteome study of Kandelia candel.

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