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.