葡萄种子蛋白质双向电泳体系的建立

Establishment of Two-dimensional Electrophoresis System of Grape Seed

为了进行葡萄种子蛋白质组学的研究,以葡萄种子为试材,通过对比粗蛋白质不同溶解方法、IPG胶条、上样量、等电聚焦参数以及不同SDS-PAGE分离胶浓度对双向电泳效果的影响,建立了适合葡萄种子的双向电泳体系。结果表明：TCA-丙酮提取蛋白质经酚抽提后蛋白质溶解快、杂质少、浓度高,筛选获得17cm pH 5～8的IPG胶条,上样量600μg,第一向的聚焦步骤达到80 000Vh以及11%的SDS-PAGE分离胶的双向电泳参数,蛋白质分离效果好。所建立的双向电泳体系,适用于不同葡萄品种的种子蛋白质分离。

Taking the grape seeds as material,in order to establish a two-dimensional electrophoresis（2-DE）system for proteomic analysis of grape seed,we compared the parameters including different protein extraction methods,the pH value gradient of IPG strip,sample loading,isoelectric focusing conditions and different SDS-PAGE separation gel.The results showed that the protein was dissolved quickly,more pure and in high concentration after extracting by TCA-acetone/phenol method.The system with 17 cm pH 5-8IPG strips,loading 600μg samples,focusing to 80 000 Vh and seperating by 11% SDS-PAGE gel showed good two-dimensional electrophoresis maps.The system was also suitable for different grape seed protein.