Comparison of Three Extraction and Purification Approaches and Optimization of Two Dimensional Electrophoresis System for Proteins from Sugarcane Leaves
为了优化适用于甘蔗叶片蛋白质的分离纯化方法与双向凝胶电泳体系,比较了3种蛋白质分离纯化方法对甘蔗叶片蛋白质的SDS-PAGE电泳图谱、蛋白质提取率以及双向凝胶电泳的影响,同时优化了双向电泳条件。结果显示,SDS提取法的SDS-PAGE电泳图谱中条带较少,蛋白质沉淀杂质多,效果差;三氯乙酸/丙酮沉淀法蛋白质沉淀得率高,但沉淀复溶性差,蛋白质溶液浓度低,难于满足双向电泳大胶的高上样量要求;酚抽提法蛋白质沉淀得率虽低,但沉淀纯度高、复溶性好,蛋白质溶液浓度高,是适合双向电泳分离纯化甘蔗叶片蛋白质的理想方法。酚抽提法蛋白质双向凝胶电泳合适的胶条p H范围为4~7,17 cm大胶合适的上样量为800μg,平均可分离出954个蛋白点。 Proteins in sugarcane leaves were extracted and purified by using three methods i.e.SDS extraction methods, trichloroacetic acid（TCA）/acetone precipitation and phenol extraction.Their productivity and profiles were assessed by means of SDS-PAGE and two-dimensional electrophoresis.The conditions of two-dimensional electrophoresis were also optimized.The results showed that the result of SDS extraction method was poor because of less bands and impurity in protein precipitation.For TCA/acetone precipitation method, the productivity of protein precipitation was high, but it was difficult to re-resolve the precipitated protein, and the resulted concentration of proteins was too low to match the normal requirement of 2-DE analysis.The phenol extraction method was found to be the best for high purification and high protein concentration.The optimized conditions for two-dimension electrophoresis of phenol extraction method was p H 4～7 for 17 cm dry strip with 800 μg protein loading amount, which produced 954 spots in average.广西自然科学基金项目（2010GXNSFA013101,2011GXNSFF018002）; 广西农科院团队项目（桂农科2011YT01
