An optimization study on two-dimensional electrophoresis for chieh-qua leaf proteomic analysis

【Objective】To establish two-dimensional electrophoresis(2-DE) system suitable for chieh-qua leaf proteomics analysis. 【Method】The effects of various protein extraction methods of 2-DE were intercompared using the leaves of “A37maojiegua” as the materials, and the loading quantity of sample，SDS-PAGE gel concentration and the pH range of IPG strips were also investigated and optimized. 【Result and conclusion】The results showed that modified phenol extraction method was notably superior to TCA/acetone precipitation method and phenol extraction method in total protein quality and clarity of protein bands in SDS-PAGE electrophoresis, and the final 2-DE image map based on modified phenol extraction method had the maxium protein dots and clear background, slight streaking, more completely separated spots compared with the other methods. The further optimization of 2-DE system was carried out to obtain a 2-DE electrophoretogram abundant protein spots, high resolution, clear background and excellent repeatability with 17 cm IPG strips,120 μg protein loading quantity, SDS-PAGE with 12% gel concentration and IPG strips with pH 4-7, finally detecting proteins with modified silver nitrate staining. More than 1 936 protein spots were detected and their relative molecular mass ranged from 15 000 to 100 000. The optimization of the experimental conditions of 2-DE system suitable for chieh-qua proteomics research has been constructed, which provides a basis for further investigation of chieh-qua proteomics researches.