文冠果枝条韧皮部蛋白质双向电泳体系的建立

Optimization of Two-dimensional Gel Electrophoresis for Proteome from the Phloem of Xanthoceras sorbifolia

以文冠果当年生硬枝韧皮部为材料,通过TCA/丙酮法提取总蛋白,研究了不同的蛋白质裂解液组成对双向电泳的影响,并在胶条选择、蛋白质上样量和等电聚焦时间等方面进行了优化。结果表明,TCA/丙酮提取法适用于提取文冠果韧皮部组织总蛋白,得到的图谱背景低,蛋白质点清晰;样品溶解于含有尿素和硫脲的蛋白质裂解液Ⅱ中,可显著地提高蛋白质的溶解性,获得的总蛋白溶液浓度远大于裂解液Ⅰ提取的总蛋白溶液浓度。在适宜的时间范围用超声波破碎仪处理蛋白质粉溶液有助于大幅度提高裂解液中蛋白质的浓度。等电聚焦条件宜根据样品特性选择,80 000Vh能够使得文冠果韧皮部蛋白质双向凝胶碱性端的横条纹大大减少。

TCA/Acetone was used for extracting total protein from the phloem of Xanthoceras sorbifolia.The effects of different lysis buffers on two-dimensional electrophoresis（2-DE） gels were compared,loading quantity of sample were primarily selected,types of strips and the time for isoelectric focusing were also optimized for the analysis of 2-DE system on the phloem.The results showed that TCA/Aceton is suitable for extracting total protein from the phloem.This method made low background and clearly protein spot on gels.Lysis buffer Ⅱ significantly increased the protein solubility;this buffer dissolved higher amounts of total protein than lysis buffer Ⅰ.Proper ultrasonic treatment in appropriate condition will significantly increase the concentration of protein solution.Isoelectric focusing conditions should be selected according to the sample characteristics,80 000 Vh was enough for isoelectric focusing in order to reduce horizontal lines on alkaline side.