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为了建立适用于水稻(Oryza sativa L.)种子蛋白质组分析的双向电泳方法,本研究比较了三氯乙酸/丙酮法和改良的酚提取法对水稻种子蛋白的提取效果,不同蛋白提取液上样量、不同p H梯度(p H 3~10和p H 4~7)IPG胶条对水稻种子蛋白的分离效果。结果表明:在目前的双向电泳体系下,采用改良的酚提取法,24 cm p H 4~7线性IPG胶条,上样量1 200~1 500μg的条件可以获得分辨率高、重复性好、胶内蛋白点适用于后续质谱分析的双向电泳图谱。最后本实验通过基质辅助激光解吸电离飞行时间质谱(MALDI-TOF/TOF-MS)对10个随机挑选的胶内蛋白质点进行了初步蛋白质鉴定分析,进一步确定了改良的酚提取法可以适用于水稻种子蛋白质双向电泳分析。
In order to establish a two-dimensional electrophoresis method suitable for the proteomic analysis of rice (Oryza sativa L.) seeds, the effects of trichloroacetic acid / acetone method and modified phenol extraction method on the extraction of rice seed protein were compared. Different protein extracts The effect of IPG strips with different p H gradient (p H 3 ~ 10 and p H 4 ~ 7) on the separation of rice seed protein. The results showed that under the current two-dimensional electrophoresis system, the improved phenol extraction method and 24 cm p H 4 ~ 7 linear IPG strip with the loading amount of 1 200 ~ 1 500 μg could obtain high resolution, good repeatability, Gel in protein spots are suitable for subsequent two-dimensional gel electrophoresis analysis. Finally, the preliminary protein identification analysis of 10 randomly selected in-gel protein spots by matrix-assisted laser desorption / ionization time of flight mass spectrometry (MALDI-TOF / TOF-MS) confirmed that the improved phenol extraction method could be applied to rice Seed protein two-dimensional electrophoresis analysis.