西藏巨柏双向电泳叶片总蛋白提取方法优化
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摘要
为了获取高质量的西藏巨柏叶片蛋白质,分别采用酚抽提法、TCA/丙酮法和改良的TCA丙酮法3种方法对西藏巨柏叶片进行总蛋白的提取,同时利用蛋白试剂盒(The 2-D Quant Kit)对蛋白质的浓度和纯度进行了分析测定,并通过蛋白质斑点数量及分布特征,最后确定了最佳蛋白提取方法。结果表明:对样品研磨的精细程度、丙酮清洗沉淀次数等操作细节均能影响提取结果,同时采用改良的TCA丙酮法提纯后的结果最佳。说明改良的TCA丙酮法可应用于西藏巨柏叶片后续的蛋白质组学研究。
In this study, to extract high-quality leaves protein of Cupressus gigantea, total leaves protein was extracted using three methods including Phenol extraction method, TCA-acetone method and modified TCA-acetone method. The concentrates and purity of the proteins were determined by The 2-D Quant Kit, and then two-dimensional gel electrophoresis was conducted in the test. The optimal method of protein extraction was determined through comparing the protein concentration, the number and distribution characteristics of protein spots. The results showed that some operational details could affect the extraction results, including the grinding degree of samples, the number of precipitation washed with acetone, and so on. The electrophoresis result was optimal through using the modified TCA-acetone method for the purification. The result indicates that the modified TCA-acetone method can be applied to the further study of leaves proteomics of Cupressus gigantea.
In this study, to extract high-quality leaves protein of Cupressus gigantea, total leaves protein was extracted using three methods including Phenol extraction method, TCA-acetone method and modified TCA-acetone method. The concentrates and purity of the proteins were determined by The 2-D Quant Kit, and then two-dimensional gel electrophoresis was conducted in the test. The optimal method of protein extraction was determined through comparing the protein concentration, the number and distribution characteristics of protein spots. The results showed that some operational details could affect the extraction results, including the grinding degree of samples, the number of precipitation washed with acetone, and so on. The electrophoresis result was optimal through using the modified TCA-acetone method for the purification. The result indicates that the modified TCA-acetone method can be applied to the further study of leaves proteomics of Cupressus gigantea.
引文
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[11]王新芳,郭娜,郭祥龙,等.萌发期大豆总蛋白提取及双向电泳体系的优化[J].中国油料作物学报, 2015,37(2):220-226.
[2]刘冰.巨柏:藏东“神木”[J].森林与人类,2016(15):64-65.
[3]王耀杰,李梓硕,刘硕,等.基于ISSR技术对西藏巨柏遗传多样性研究[J].辽宁林业科技, 2017(6):12-14.
[4]傅立国.中国珍稀濒危植物的福音-国家重点保护植物名录公布在即[J].植物杂志,1995(3):2-3.
[5]郭其强,李慧娥,张艳福,等.不同浓度ABT和浸泡时间对西藏2种柏树扦插生根的影响[J].中南林业科技大学学报,2018,38(7):14-18.
[6]龚迪,毕阳,王军节,等.基于蛋白组学的水果蛋白质提取方法[J].食品工业科技,2014,35(3):376-379.
[7]庞新华,张宇,黄国弟,等.4种黄皮叶片蛋白质提取方法的比较[J].经济林研究,2017,35(1):59-63.
[8]曹原,周宇航,王忠奎,等.光核桃叶片蛋白双向电泳技术的完善[J].黑龙江畜牧兽医, 2017(5):129-131.
[9]龚迪,毕阳,王军节,等.基于蛋白组学的水果蛋白质提取方法[J].食品工业科技,2014,35(3):376-379.
[10]王珊珊,郝宇涵,任婧祺,等.黄瓜根系蛋白质组分析的双向电泳技术条件优化[J].沈阳农业大学学报,2012,43(2):233-236.
[11]王新芳,郭娜,郭祥龙,等.萌发期大豆总蛋白提取及双向电泳体系的优化[J].中国油料作物学报, 2015,37(2):220-226.