箭毒木种子蛋白质样品制备及双向电泳改良方法

The Sample Preparation and the Improvement Method of Two-Dimensional Electrophoresis of Proteins from Antiaris toxicaria (Moraceae) Seed

建立箭毒木(Antiaris toxicaria)种子总蛋白的提取方法,以及可以对其蛋白质组进行分析的双向电泳条件。通过各种条件的优化与组合,建立了以TCA-丙酮为基础的Tris-HCl提取法提取总蛋白,第1向电泳为固相pH梯度等电聚焦,第2向电泳为垂直平板SDS-PAGE的双向电泳体系。通过对样品制备、样品溶解、等电聚焦电泳、SDS——聚丙烯酰胺凝胶电泳以及染色方法等关键步骤进行分析,获得了满意的双向电泳图谱。在探索适合箭毒木种子蛋白质组学研究双向电泳方法中,比较了三氯乙酸-丙酮沉淀法、和Tris-HCl法,以及对双向电泳过程中的关键步骤的改良,认为Tris-HCl法为最适方法,所得图谱背景清晰,蛋白质信息量最大,为箭毒木属植物的差异蛋白质组学的后续研究打下了坚实的基础。

We have established the extraction method of the total proteins of Antiaris toxicaria seed and the appropriate conditions for the two-dimensional eiectrophoresis （2-DE） of Antiaris toxicaria seed proteome. Through integrating and optimizing various conditions, we also established the 2-DE system including the sample preparation method with Tris-HCL based on TCA-actone, and the first-dimensional electrophoresis with immobilized pH gradients isoelectric focusing and the second dimensional electrophoresis with vertical electrophoresis of SDS-PAGE. We have gained a good 2-DE map in the analysis of sample preparation, sample resolution, isoelectric focusing （IEF）, SDS-PAGE and gel staining. In order to explore the extraction method of proteome and technique of 2-DE, we compared the extraction method between the TCA-actone and the Tris-HCl, and improved the key steps of 2-DE. The Tris-HCl extraction was the most appropriate for Antiaris toxicaria seed proteome analysis because of the highest resolution and more informative 2-DE map. Those were the solid basis for the later research of studying on the discrepancy expression proteomics of Antiaris toxicaria Seed.