当归种子蛋白质提取方法及SDS-PAGE技术体系研究
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篇名: 当归种子蛋白质提取方法及SDS-PAGE技术体系研究
TITLE:
摘要: 目的:建立适用于当归种子蛋白质提取及十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)的技术体系,为当归种子蛋白质研究和品种纯度检测提供技术支撑。方法:以蛋白质含量和电泳条带数量等为指标,采用浓缩胶法、盐溶蛋白法、电极缓冲液法、二巯基苏糖醇(DTT)法、尿素法、巯基乙醇法、三羟甲基氨基甲烷(Tris)法、丙酮沉淀法等8种方法提取当归种子蛋白质,筛选最优提取方法;再以最优提取方法为基础,考察不同料液比、样品稀释倍数(上样量)和分离胶浓度对当归种子蛋白质提取及SDS-PAGE效果的影响。结果:8种提取方法中以巯基乙醇法提取蛋白质的含量较高(29.931 mg/g),且电泳条带数目多、泳道背景清晰;当以巯基乙醇法为最优提取方法时,料液比为1 ∶ 10、上样量为5倍、分离胶浓度为15%条件下电泳效果最好,共得到18条条带。结论:建立的蛋白质提取方法和SDS-PAGE技术体系适用于当归种子纯度的检测。
ABSTRACT: OBJECTIVE: To establish the technical system that is suitable for protein extracting in Angelicae sinensis seed and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and provide technical support for detecting the protein quality and variety  purity. METHODS: Using protein content and number of electrophoretic bands as indexes, 8 methods, including voncentrated gel method, salt-soluble protein method, electrode buffer method, dimercaptosyl alcohol (DTT) method, urea method, mercaptoethanol method, trimethylolaminomethane (Tris) method, and acetone precipitation method, were conducted to extract the protein in A. sinensis seed and screen the optimal extraction method. Then based on optimal extraction method, effects of different materials-lipid ratios, sample dilution times (sample volume) and separate gel concentration on SDS-PAGE were investigated. RESULTS: Mercaptoethanol method extracted the highest protein contents (29.931 mg/g), with many electrophoretic bands and clear background. When mercaptoethanol method was used as optimal extraction method, electrophoresis effects were the best in the conditions of materials-lipid ratio of 1 ∶ 10, sample volume of 5 times, separate gel concentration of 15%, which obtained 18 bands totally. CONCLUSIONS: Established protein extraction method and SDS-PAGE technical system are suitable for detecting the purity of A. sinensis seed.
期刊: 2017年第28卷第13期
作者: 张延红,高素芳,王燕,李金田,杜弢
AUTHORS: ZHANG Yanhong,GAO Sufang,WANG Yan,LI Jintian,DU Tao
关键字: 当归;种子;蛋白质提取;SDS-PAGE
KEYWORDS: Angelicae sinensis; Seed; Protein extraction; SDS-PAGE
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