西瓜根系蛋白质双向电泳体系的建立及优化

Establishment and Optimization of Two-dimensional Electrophoresis System of Watermelon Root

为建立高效、稳定的西瓜根系蛋白质双向电泳技术体系,对西瓜根系蛋白质样品等电聚焦(IEF)条件、样品上样量、IPG胶条p H值范围、染色方法等参数进行优化。结果表明,采用TCA/丙酮法提取西瓜根系总蛋白,裂解液组成为7 mol/L Urea、2 mol/L Thouirea、4%CHAPS(m/V)、1%Cocktail(V/V)、65 mmol/L DTT、2%p H值4~7 IPG Buffer,上样量为800μg,p H值4~7 IPG(13 cm)胶条、IEF条件为48 000 Vh,分离胶浓度为12.5%,CBB-R250染色时,可获得分辨率高、重复性好的双向电泳图谱,该技术条件为适合西瓜根系蛋白分离的较优双向电泳体系。

An efficient and suitable two-dimensional electrophoresis(2-DE) system for proteomic analysis of watermelon roots was established by optimizing the isoelectric focusing conditions,sample loading,the pH gradient of IPG strips and staining methods.Results showed that the optimized system for watermelon roots were as following steps:total protein of watermelon roots was extracted by TCA/acetone precipitation,lysis Buffer contained 7 mol/L Urea,2 mol/L Thouirea,4% CHAPS(m/V),1% Cocktail(V/V),65 mmol /L DTT and 2% pH 4 -7 IPG Buffer, 13 cm pH 4 -7 IPG strips was used to separate the proteins,loading protein samples was 800 μg,IEF condition was 48 000 Vh,12.5% gel concentration and followed by staining with colloidal Coomassie Brilliant Blue R-250.