奶牛乳腺组织亚细胞组分的双向凝胶电泳分析

利用超速离心结合梯度离心法分离亚细胞组分的技术路线,以提高奶牛乳腺组织蛋白双向凝胶电泳的分离效率。奶牛乳腺组织液氮研磨破碎后,差速离心分离成细胞核、线粒体、高尔基体、溶酶体4个亚细胞组分,Nyco-denz纯化,2-DE分离蛋白,PDQUest8.0软件分析凝胶图谱。结果显示,奶牛乳腺组织细胞经亚细胞分离后,电泳分辨率大大提高,特别是细胞核蛋白质检出效率明显提高。不同亚细胞蛋白质组电泳图谱的差异显示了其蛋白质组构成的不同。由此可见超速离心结合梯度离心是一种有效的奶牛乳腺亚细胞组分分离手段,亚细胞蛋白质组学克服了蛋白质组的一些缺陷并可将蛋白质组研究引向亚细胞水平。

Analysis of two-dimensional Gel electrophoresis of sub-cellular fractions of mammary gland from dairy cows

To improve the separation efficiency of 2-DE in proteins from mammary gland in dairy cows,ultracentrifuge combined with gradient centrifuge were used in separating sub-cellular components.The homogenated mammary gland was fractionated into nucleis,mitotrondria,Golgi apparatuses,lysosomes four parts by differential centrifugation and density gradient centrifugation,proteins extracted from four fractions is subjected to two-dimensional Gel electrophoresis,and the 2-DE patterns are analyzed by PDQUest 8.0 software.In comparision with the 2-DE patterns of total proteins,the patterns of the four fractions each display more protein spots,and a large number of proteins present in the nuclei and were not showed in the not-fractionated sample.So,preparation of sub-cellular fractionation before electrophoresis procedures were proved to be very useful,not only improve the separation efficiency of two-dimensional Gel electrophoresis,but also overcome some defects of two-dimensional Gel electrophoresis,and lead proteome to sub-cellular level.