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为了获得甜菜叶片双向电泳中蛋白质最佳提取方法,建立甜菜叶片蛋白质双向电泳最佳的双向电泳体系。以甜菜叶片为材料,通过比较分析,研究甜菜叶片总蛋白提取方法、IPG胶条pH值及其对应上样量等因素对甜菜叶片蛋白质双向电泳结果的影响。结果表明:酚提取法、TCA/丙酮提取法和可溶性蛋白提取法3种蛋白提取方法中TCA/丙酮法提取效果较好,pH值4~7与pH值5~8的双向蛋白电泳比较发现:pH值4~7胶条蛋白点清晰且在图谱上的蛋白点分布均匀,无蛋白点集中现象,更适合甜菜叶片的蛋白质组分析。采用7 cm线性固相IPG胶条进行双向电泳,150μg上样量蛋白点明显增多,双向电泳效果更好,pH值4~7的17 cm胶条300μg上样量胶点更多更清晰,试验结果为甜菜蛋白质组学的研究提供了最佳的试验方法。
In order to obtain the best extraction method of protein in two-dimensional electrophoresis of beet leaf, two-dimensional electrophoresis system of two-dimensional electrophoresis of beet leaf protein was established. In this paper, the effects of extraction methods of total protein in beet leaves, pH value of IPG strips and the corresponding loading amount on the two-dimensional electrophoresis results of beet leaf protein were studied by comparative analysis. The results showed that TCA / acetone extraction method was better than phenol extraction method, TCA / acetone extraction method and soluble protein extraction method. The comparison of two-way protein electrophoresis between pH 4 ~ 7 and pH 5 ~ 8 showed that: The protein spots with pH value of 4 ~ 7 were clear and the protein spots on the map were evenly distributed, and no protein spots were concentrated, which was more suitable for the proteome analysis of beet leaf. Using 7 cm linear solid phase IPG strip for two-dimensional electrophoresis, the amount of protein spots on the 150 μg sample increased obviously, and the two-dimensional electrophoresis effect was better. The gel spots of 300 μg on the 17 cm strip with pH value of 4 to 7 were more and more clear. The results provide the best experimental method for the study of beet proteomics.