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.

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