东北龙胆花芽蛋白质双向电泳条件的建立

Establishment of A Two Dimensional Electrophoresis Protocol for Protein of Gentiana manshurica Flower Bud

初步建立了东北龙胆(Gentiana manshuica Kitagawa)花芽总蛋白质提取方法,以及东北龙胆花芽蛋白质组研究的双向电泳技术。从样品制备及溶解等方面进行了优化,解决了东北龙胆花芽蛋白质提取中色素、多糖、酚类物质等对双向电泳的干扰,确定了一种有效的蛋白溶解液(7mol/L尿素,2mol/L硫脲,4%CHAPS,40mmol/LDTT,1%蛋白酶抑制剂混合物,2%的两性电解质pH=3～10)。同时,对电泳其它环节也进行了优化,经考马斯亮兰染色后,可分辨蛋白质斑点数约1000个。获得了重复性好、分辨率高的蛋白质双向电泳图谱。

An extraction method of total protein from Gentiana manshurica Kitag. flower bud and the technology of two-dimen-sional electrophoresis of flower bud proteome were initially established in order to raise resolution and repeatability. The preparation and dissolution of samples were optimized, which solved the interference of pigment, polysaccharide, and phenol materials to the two-dimensional electrophoresis disturbance in the process of extraction of G. manshurica flower bud. A kind of effective protein dissolution fluid was determined as 7 mol/L urea, 2 mol/L thiourea, 4% CHAPS, 40 mmol/L DTr, and 2% ampholyte pH 3-10. The optimization of other links in electrophoresis was also conducted. After the Coomassie Brilliant Blue staining, it could distinguish approximately 1000 protein spots. A two-dimensional electrophoresis atlas with good repeatability and high resolution was obtained.