产热凝胶土壤杆菌ATCC31749蛋白质组双向电泳图谱的构建

建立了热凝胶生产茵土壤杆茵茵体总蛋白的蛋白质提取方法和双向电泳方案,确定了使用蛋白质裂解液(7 mol/L尿素,2 mol/L硫脲,1%ASB-14去垢剂,40 mmol/L Tris,0.001%溴酚蓝,1 mmol/L EDTA,1%TBP和1%两性电解质)结合超声破碎法来提取茵体总蛋白的方案为最佳,选择17 cm...

Construction of two-dimensional electrophoresis (2-DE) proteomic map of Agrobacterium species ATCC31749

The method for extraction total cellular protein of Agrobacterium species for 2-DE was constructed and optimized. The optimum process was the following： cells suspended in 1 mL of a lysis buffer （7 mol/L urea, 2 mol/L thiourea, 1% ASB-14 detergent, 40 mmol/L Tris base, 0. 001% bromophenol blue, 1 mmol/L EDTA, 1% TBP and 1% carrier ampholyte）, and then sonication for 3 min on ice. After centrifugation, the supematant was reserved in -80℃ for two-dimensional electrophoresis （2-DE）. And the 2-DE conditions were also constructed. The immobilized pH gradient （IPG） gel strips （17 cm） with linear pH 4- 7 were used for the first dimension electrophoresis with suitable electrophoresis parameters. After compared two staining methods （coomassie blue staining and silver staining）, it was found that the silver staining could get more and clearly protein spots on 2-DE map. Finally, the method for constructing 2-DE protein map with high resolution rate and good repeatability was established.