棉花学报2012，Vol.24Issue(1)：27-34,8.
棉花叶片双向电泳体系的研究
Development of Two-dimensional Electrophoresis Protocol Suitable for Proteomic Analysis of Cotton Leaves
摘要
Abstract
We have developed a two-dimensional gel electrophoresis method to analyze the proteomics of cotton Gossypium hir-sutum L. Leaves. We investigated and compared five processes for the extraction of total proteins from cotton leaves. Two different ranges of a linear immobilized pH gradient(IPG), four different sample loading, reductant gradients and carrier ampholytes in IPG buffer solution were compared. A high purity of cotton protein was extracted by TCA-acetone precipitation, and by cleaning the protein sample with solvent methanol or acetone. The parameters for obtaining a high resolution of cotton 2-D electrophoresis maps were optimized as follows: 24 cm strip with linear (IPG) in pH 5-8, 300 |xg sample loading, Tributyl phosphate(TBP) substituting for DL-Dithiothreitol (DTT) in the IPG buffer, and a combination of two carrier ampholytes in the pH range of 5-8 and 3-10, with a volume ratio of 1 : 1. This optimal 2-D electrophoresis system gave a higher resolution, protein yield, and spot intensity in 2-D electrophoresis analysis.关键词
棉花叶片/蛋白质提取方法/蛋白组学/双向电泳Key words
cotton leaves/ protein extraction/ proteomics/ two-dimensional gel electrophoresis分类
农业科技引用本文复制引用
韩吉春,崔海峰,时鹏涛,祝水金,叶子弘,俞晓平..棉花叶片双向电泳体系的研究[J].棉花学报,2012,24(1):27-34,8.基金项目
农业部转基因重大专项(2009ZX08012-013B) （2009ZX08012-013B）
杭州市科技局重点项目(20101032B28) （20101032B28）
