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

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.

Key words:radiation-resistance bacterium; Micrococcus luteus; protein extraction; two-dimensional gel electrophoresis