肺炎链球菌蛋白质组双向电泳技术的建立及优化

为了建立适合肺炎链球菌(Streptococcus pneumoniae)蛋白质组的双向电泳技术,对蛋白样品提取、裂解、上样量、IEF和银染等关键步骤进行了优化,探索出一套有效的肺炎链球菌蛋白分析的双向电泳方法,即以裂解液15 mmol/L Tris-HCl、7 mol/L尿素、2 mol/L硫脲、4%(m/v)CHAPS,使用前现加2%(v/v)IPG缓冲液、1%(m/v)二硫苏糖醇(DTT)和1%(m/v)蛋白酶抑制剂混合物]结合反复冻融和超声裂解菌体的方法来提取蛋白质样品,按80μg蛋白上样量,并结合适宜的双向电泳参数,可获得蛋白分辨率高、重复性较好的双向电泳图谱。

Establishment and Optimization of Technique of Two-dimensional Electrophoresis for Proteome of Streptococcus pneumoniae

In order to establish an efficient two-dimensional gel electrophoresis(2-DE) protocol for proteomic study of Streptococcus pneumoniae,the processes of cell lysis,protein extraction,protein loading,IEF and silver staining were optimized.It was found that the following treatments could obtain 2-DE protein map with high resolving rate and good repeatability: cells were treated consecutively with a lysis buffer containing 15 mmol/L Tris-HCl,7 mol/L urea,2 mol/L thiourea,1% DTT,4% CHAPS,2%(v/v) IPG buffer with p...