东方伊萨酵母降解染料蛋白质组的双向电泳条件优化

利用双向凝胶电泳技术分离东方伊萨酵母Issatchenkia orientalis降解染料活性艳红K-2BP前后细胞内总蛋白质,并比较不同样品处理方法、不同上样量以及不同染色方法等对电泳结果的影响,进而获得较优的分离染料降解相关蛋白的双向凝胶电泳条件.在3种样品处理方法中,单纯超声波破碎,超声波-TCA/丙酮沉淀,超声...

Optimization of Two-dimensional Electrophoresis Conditions for Proteomics of Issatchenkia orientalis in Degrading Dyes

Total protein of the yeast Issatchenkia orientalis was extracted and separated by two-dimensional electrophoresis (2-DE) before and after the dye Reactive Brilliant Red K-2BP was degraded by this yeast, respectively. Different protein extraction methods, different volumes of sample loaded and different staining techniques were tested and compared for the 2-DE. Among three different protein extraction methods, the final protein concentrations of 3.4 mg/mL, 1.8 mg/mL and 5.6 mg/mL were obtained by single ultrasonication, ultrasonication-TCA/acetone,and ultrasonication-ammonium sulfate precipitation, respectively. The best electrophoresis pattern could be gotten by loading 150 microg protein samples from the method of ultrasonication-ammonium sulfate precipitation, using IPG strips of pH 4-7 for the first dimensional electrophoresis and staining with silver nitrate. This electrophoresis pattern had high resolution and good repetition. It was detected to have 730 +/- 30 protein points by preliminary image analysis. This research results provided a technical support for screening dye-degrading enzymes from the yeast of I. orientalis.