乳小鼠培养肝细胞蛋白质双向电泳技术的建立

PRELIMINARY ESTABLISHMENT OF TWO-DIMENSIONAL ELECTROPHORESIS FOR PROTEOMICS OF CULTURED HEPATOCYTE OF NEw-BORN MICE

目的 :建立培养乳小鼠肝细胞蛋白质的双向电泳技术。方法 :取乳小鼠肝脏组织 ,进行肝细胞原代培养 ,提取肝细胞蛋白 ,以固相pH梯度 (IPG)等点聚焦为第一向 ,垂直SDS -PAGE为第二向进行双向电泳 ,并对样品处理方式、蛋白上样量、IPG等电聚焦和SDS -PAGE电泳参数设置、凝胶浓度等关键因素和环节进行了比较研究。结果 :通过实验条件的筛选和优化获得了较满意的双向电泳图谱。结论 :本文建立乳小鼠双向电泳分离技术 ,具有较高的分辨率和重复性 。

Objective: To establish and optimize the two-dimensional electrophoresis (2-DE) for proteins in cultured hepatocyte of new-born mice. Method: Hepatocytes of new-born mice were primary cultured. The proteins were extracted by a lysis buffer containing 8M urea, 4%CHAPS, 2%Bio-lyte(pH4～7), 40mM TBP. One hundred micrograms of protein was loaded on 17cm IPG strip holder and run isoelectric focusing electrophoresis as the first dimension, then vertical SDS-PAGE as the second dimension. A series of important factors, such as sample handling, gel preparation, electrophoresis parameters and so on were optimized. Result: The 2-DE patterns with good feature have been obtained. Conclusion: With optimization of each condition, two-dimensional electrophoresis (2-DE) has been preliminarily established.