金华火腿品质的特征蛋白质及其变化研究--金华火腿双向电泳样品制备研究

Research on Jinhua Ham Characteristic Proteins and Their Changes——Research on sample preparation of Jinhua ham for two-dimensional electrophoresis(2-DE)

为寻找针对金华火腿肌肉组织的双向电泳样品处理方法,文中分别采用组织裂解液法、TCA/丙酮沉淀法、蛋白纯化试剂盒法、TCA/丙酮沉淀-蛋白纯化试剂盒法、凝胶层析-冷冻干燥-裂解法5种方法进行样品制备实验,上样量为300μg蛋白质,以17cmpI5～8固相pH梯度胶条做第一向等电聚焦,SDS-PAGE垂直电泳为第二向进行双向电泳(2-DE),银染,图像分析软件分析电泳图谱。结果显示:利用凝胶层析-冷冻干燥-裂解法提取的金华火腿肌肉组织蛋白2-DE凝胶图像纵条纹、横条纹以及背景都明显减少,获得蛋白点数量较其他4种方法增加,同时用此种方法获得的样品在等电聚焦时比较稳定。

To find out a suitable method for refining protein of Jinhua ham muscle tissue, lysate method, TCA / acetone precipitation method, 2-DE clean-up kit, TCA / acetone and clean-up kit, gel column chromatography method were used. In the 2-DE system,17 cm pI 5 - 8 IPG gel was chosen as the first dimension of isoelectro-focusing electrophoresis; the second dimension was SDS-polyacrylamide gel electrophoresis,300ug protein was segregated and the 2-DE maps were obtained. The electropherogram were analyzed by using image analysis software. The Results showed the horizontal stripes, vertical stripes, and background of the 2 - DE maps of protein refined by gel column chromatography method decreased obviously. The protein spots obtained by gel column chromatography method increased significantly. At the same time the samples obtained by gel column chromatography method were more stable in isoelectric focusing. This may provided a foundation for the further study of the Jinhua ham characteristic protein and its changes.