大鼠肾脏组织蛋白质组双向电泳分离体系的建立

目的:建立大鼠肾脏组织蛋白质组双向电泳分离体系。方法:提取大鼠肾脏组织的样品蛋白,按标准条件对蛋白质进行双向电泳分离,并对各个关键因素进行优化。结果:最终选择的裂解液配方是1%TBP,4%CHAPS,0.2%Bio-Lyte,40mmol/LTris,8mol/L尿素,2mol/L硫脲;使用pH 4～7的IPG胶条进行被动水化上样,等电聚焦采用缓慢升压模式,电泳参数根据Bio-Rad公司的预设方案进行调整;改良硝酸银法进行蛋白质斑点染色。从而获得了满意的蛋白质组双向电泳图谱。结论:成功建立具有较高分辨率和重复性的大鼠肾脏组织蛋白质组双向电泳分离体系,为后续实验研究提供有力的技术保障。

ESTABLISHMENT OF TWO-DIMENSIONAL ELECTROPHORESIS SYSTEM FOR PROTEOMES FROM RAT KIDNEY TISSUE

Objective:To establish a system of two-dimensional gel electrophoresis(2-DE) for separation of proteomes from rat kidney tissue.Methods:Proteins were extracted from rat kidney tissue after their lysing,and separated by 2-DE in standard conditions,and important experimental parameters were optimized.Results:The lysis buffer containing 1%TBP,4%CHAPS,0.2%Bio-Lyte,40 mmol/L Tris,8 mol/L urea,and 2 mol/L thiourea was applied to extract proteins from tissues.The passive rehydration scheme of samples was used to help the separation of large molecular weight proteins in the IPG gel.Electrophoresis parameters recommended by Bio-Rad Corporation were adjusted adequately.In the process of silver staining,a sufficient washing operation ensured a weak background of 2-DE maps.Conclusion:Under above optimized conditions,the 2-DE separation system for proteomes from rat kidney tissue are successfully established,which will guarantee the further exparimental study.