耐辐射菌Micrococcus luteus SC1204总蛋白双向电泳体系的建立

Optimization of two-dimensional gel electrophoresis (2-DE) system for protemics analysis of a radiation-resistant bacterium Micrococcus luteus SC1204

摘要:

以耐辐射藤黄微球菌Micrococcus luteus SC1204为研究对象，比较蛋白质提取方法、蛋白质裂解液配方、IPG胶条pH梯度、蛋白上样量及等电聚焦时间对M. luteus SC1204总蛋白双向电泳的影响。结果表明，采用液氮研磨—酚/超高速离心法提取总蛋白，裂解液Ⅱ(8 mol/L尿素、 2 mol/L硫脲、60 m mol/L DTT、4% CHAPS、40 m mol/Ltris、1% pH 3-10 NL IPG buffer、0.002% BPB)溶解蛋白，使用24 cm、pH 4-7的IPG胶条，上样量250 ug及等电聚焦7 h ( 56000 Vh )，可获得满意分辨率的双向电泳图谱，适用于后续的质谱及差异蛋白质组分析。

Abstract:

The total proteins extracted from a radiation-resistant bacterium Micrococcus luteus SC1204 were separated by immobilized pH gradient-based two-dimensional gel electrophoresis (2-DE), and ImageMaster 2D Platinum 5.0 was applied to analyse 2-DE images after silver staining. The 2-DE was optimized by comparative tests on the important factors including extraction methods, lysis buffer components, pH range of IPG strips, sample volume, and isoelectric focusing time. The results showed that the resolution of 2-DE profiles was significantly improved by liquid nitrogen grinding-phenol∕ultracentrifugation in lysis buffer Ⅱ for sample preparation, pH 4-7 (24 cm) IPG gel strips, the sample loading at 250 μg, and prolonged isoelectric focusing time (56000 vhr). This work provided a technical basis for the further study on differential proteomics in M. luteus SC1204.