【目的】比较鱼腥草叶片蛋白质提取方法,建立适用于鱼腥草蛋白质组双向电泳分析方法。【方法】以鱼腥草叶片为材料.分别采用凯基试剂盒、三氯乙酸/丙酮沉淀法、尿素/硫脲提取法及酚法4种方法提取鱼腥草叶片总蛋白,使用Bradford法测定蛋白质浓度,计算蛋白提取率.并对所提取的蛋白样品进行聚丙烯酰胺凝胶电泳(SDS-PAGE)及双向电泳(2-DE)分析,所得2-DE凝胶图片采用PDQuest软件进行分析。【结果】4种方法的提取率分别为2.90、2.55、3.90、3.80mg/g,分别检测到186、153、356、315个蛋白点,酚法提取蛋白质的2-DE图谱效果较好,结果的重复性较好。【结论】不同方法提取鱼腥草叶片总蛋白的2-DE结果存在差异,以酚法提取的2-DE效果较好,适用于鱼腥草叶片蛋白质组分析。
Objective To establish two-dimensional electrophoresis (2-DE) proteome analysis method for total protein from the leaves of Houttuynia cordata Thunb. Methods The four methods, including Keygen kit extraction (A), trichloracetic acid/acetone precipitation (B), urea/thiourea extraction (C), and phenol extraction (D), were adopted to extract the total protein from the leaves of Houttuynia cordata Thunb. The protein concentration was determined by Bradford protein assay for calculation of the protein yield, and then the protein samples were separated by sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) and 2-DE. The 2-DE gel images were analyzed by PD Quest software. Results The protein yield by the method A, B, C and D was 2.90, 2.55, 3.90 and 3.80 rag/g, respectively, and 186, 153, 356 and 315 spots could be detected separately in 2-DE gel electrophoretograms of the four methods. Conclusion Different 2-DE results of total protein extracted from the leaves of Houttuynia cordata Thunb have been achieved by the four methods, and the phenol extraction method presents satisfactory results, better than the others.