苹果叶片总蛋白提取及其双向电泳分析

蛋白质提取方法和电泳条件的建立是蛋白质组学研究中双向电泳分析的关键。通过比较不同提取方法,包括酚/SDS抽提、TCA-丙酮沉淀法、甲醇/醋酸铵沉淀法以及改良HB-酚抽提方法。此外,还优化了样品上样量、等电聚焦程序设置等条件。从所得2-D图谱分离效果来看,改良HB-酚抽提方法所得蛋白点数最多,2-D图谱背景清晰、分离效果好,明显优于其他3种方法。300μg上样量可作为苹果叶片总蛋白2-DE分析的理想的上样量,同时优化了等电聚焦程序,最终得到的2-D图谱分离效果较为理想。该研究的开展也为苹果叶片蛋白质组学后续研究奠定了基础。

Extraction and two-dimensional gel electrophoresis analysis of total protein from apple leaves

The optimal protein extraction methods and electrophoresis conditions are the key factors of two-dimensional electrophoresis(2-DE) in proteomics.In this study,in order to establish a suitable method for the total protein of apple leaves,and to optimize the conditions for 2-DE,we compared different protein extraction methods including Phenol/SDS extraction,TCA-acetone precipitation,methanol/ammonium acetate precipitation,and improved HB-Phenol extraction.We also optimized the sample loading quantity and the key process of IEF.The results showed that the improved HB-Phenol extraction protocol was the best for apple leaves,which can obtain more protein spots,distinctly background and better separation efficiency.The optimal sample loading quantity was 300 μg,which was suitable for the 2-DE analysis..An ideal separation was also obtained by optimizing the procedure of IEF.The study built up a good basis for the future proteomics analysis of apple leaves.