福建柏叶片蛋白质双向电泳技术优化

Protocols optimization in two-dimensional electrophoresis of Fokienia hodginsii leaves total protein

蛋白质双向电泳是蛋白质组学研究方法之一,但蛋白质双向电泳技术体系却因物种不同而有所差异,因而蛋白质双向电泳体系的建立是进行蛋白质分离、鉴定的首要步骤。本文对福建柏叶片蛋白质提取方法、IPG胶条、等电聚焦参数设定、丙烯酰胺分离胶浓度等因子进行单因素分析,对福建柏双向电泳技术进行优化。结果表明,采用三氯乙酸-丙酮提取福建柏叶片的蛋白点多且图像清晰,p H4~7的IPG胶条最适合福建柏叶片蛋白的分离,等电聚焦参数设定为20 000 Vh,浓度为10%的分离胶进行福建柏双向电泳最好。本研究初步建立了福建柏叶片蛋白质双向电泳体系,为深入研究福建柏蛋白质组学提供依据。

In order to study the most efficient 2-DE protocol of Fokienia hodginsii proteomics,three different methods including TCA-acetone,phenol extraction method and TCA-phenol extraction method were tested in the experiment. Besides,three different IPG strips of p H3 - 10,p H5 - 8,p H4 - 7 combined with three different focusing parameter which showed 20 000,25 000,30 000 Vh respectively were performed in IEF. Finally,different concentration separation gel（10%,12.5%） were performed in all SDS-PAGE trial. The results indicated that more protein spots were detected on 2-DE gels by phenol extraction method,IPG strips of p H4 -7,25 000 Vh focusing parameter,and 10% separate gel were the best settings of the 2-DE protocol. The experiment preliminary established the 2-DE protocols of F. hodginsii leaves which would be beneficial for the further study of proteinmics.