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目的探讨超滤法和7种不同沉淀方法纯化含PBS的蛋白样品对蛋白凝胶电泳的影响。方法应用超滤法和7种沉淀方法[包括硫酸铵、氯仿甲醇、酸化丙酮甲醇、乙醇、丙酮、三氯乙酸(TCA)、TCA/丙酮]纯化PBS稀释的混合食管癌组织蛋白(10例),二喹啉甲酸(BCA)、Bradford及2D法测定蛋白浓度并进行一维和二维聚丙烯酰胺凝胶蛋白电泳,Image Master软件分析二维电泳图谱蛋白点。结果 Bradford法和2D法适用于二维电泳水化液溶解的蛋白样品浓度测定。超滤法和硫酸铵沉淀法的蛋白回收率分别为40%和4%,其他方法的蛋白回收率约为22%。丙酮沉淀法纯化蛋白的二维电泳图谱蛋白点边界清晰、分辨率高且数目最多;其次是乙醇沉淀和氯仿甲醇沉淀法;再其次是超滤法,该法纯化蛋白的二维电泳图谱蛋白点表现不同程度的弥散;酸化丙酮甲醇沉淀蛋白的二维电泳图谱中蛋白点丢失最多。结论丙酮沉淀法适用于原始浓度高、体积小的蛋白样品除盐及浓缩,超滤法是原始体积较大、浓度较低蛋白样品浓缩纯化的首选方法。
Objective To investigate the effect of ultrafiltration and seven different precipitation methods on protein gel electrophoresis (PAGE). Methods Mixed esophageal cancer tissue protein (10 cases) diluted in PBS was purified by ultrafiltration and seven precipitation methods [including ammonium sulfate, chloroform methanol, acidified acetone methanol, ethanol, acetone, trichloroacetic acid and TCA / , Bicinchoninic acid (BCA), Bradford method and 2D method. One-dimensional and two-dimensional polyacrylamide gel electrophoresis were performed. Image Master software was used to analyze two-dimensional electrophoresis protein spots. Results The Bradford method and the 2D method were applied to the determination of protein concentration in two-dimensional electrophoresis hydration solution. The protein recovery by ultrafiltration and ammonium sulfate precipitation was 40% and 4%, respectively. The protein recovery by other methods was about 22%. The two-dimensional electrophoresis pattern of protein purified by acetone precipitation has clear and high-resolution protein spots, followed by ethanol precipitation and chloroform precipitation, followed by ultrafiltration. The two-dimensional electrophoresis protein spots Showing different degrees of dispersion; acidic acetone acetone precipitated protein two-dimensional electrophoresis map protein spots lost the most. Conclusion The acetone precipitation method is suitable for the desalination and concentration of protein samples with high original volume and small volume. Ultrafiltration is the preferred method for the concentration and purification of original and relatively concentrated protein samples.