摘要
蛋白质提取方法和电泳条件是蛋白质组学研究中双向电泳的关键。为建立适合甘蓝柱头总蛋白质的双向电泳体系,本试验以甘蓝柱头为材料,采用改良TCA/丙酮沉淀法提取柱头总蛋白质,通过对IPG胶条、上样量、聚焦程序、SDS-PAGE胶浓度等的优化,建立了甘蓝柱头总蛋白质双向电泳体系,即选用长17cm、pH3~10NL的IPG胶条,上样量150μg,按照聚焦程序Ⅰ进行等电聚焦,第二向SDS-PAGE胶浓度选用12%,得到了分辨率高、重复性好的双向电泳图片。
A two-dimensional electrophoresis(2-DE)has been widely applied in proteomics research.The purpose of this study was to establish a suitable 2-DE system for proteomic analysis of Brassica oleracea L.var.capitata L.stigma.The stigmas being harvested one day before flowering was used in the experiment.We optimized the parameters of 2-DE including extraction methods,IPG strip,sample loading,isoelectric focusing conditions and SDS-PAGE gel concentration.The optimized system included the following steps:extracting the total proteins from Brassica oleracea stigma by TCA/acetone precipitation,separating the proteins with length 17 cm and pH 3-10 NL IPG strips,loading proteins samples of 150 μg followed by isoelectric focusing programⅠ,and selecting SDS-PAGE gel concentration at 12%.The 2-D images with good repeatability and high protein spot resolution were obtained.
出处
《中国蔬菜》
北大核心
2012年第06X期30-36,共7页
China Vegetables
基金
国家自然科学基金项目(30900986
31071802)
重庆市自然科学基金项目(2009BB1298)
西南大学博士基金项目(SWUB2008042)
中央高校基本科研业务费专项(XDJK2010B010)
关键词
甘蓝柱头
自交不亲和
蛋白质双向电泳
Brassica oleracea L.var.capitata L.stigma
Self-incompatibility
Protein two-dimensional electrophoresis