建立人肾小管上皮细胞蛋白质组双向电泳的分离体系

背景：双向电泳分离技术是蛋白质组学研究的核心技术之一，但蛋白质样品的分离效果受各种实验条件的影响较大。因此，针对不同来源的蛋白样品进行实验条件的优化可获得具有较高分辨率的双向电泳图谱。目的：拟建立优化的人肾小管上皮细胞株蛋白质组双向电泳分离体系。方法：常规培养人肾小管上皮细胞株HK-2细胞并裂解提取全蛋白，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。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。采集电泳图谱并分析双向电泳图谱中蛋白斑点的数量、图像分辨率及背景条纹的变化。结果与结论：通过对实验条件的筛选和优化，成功建立了具有较高的分辨率和重复性的人肾小管上皮细胞蛋白质组双向电泳分离体系。其中，优化后的裂解液配方成分为1% TBP，4%CHAPS，0.2% Bio-Lyte，40 mmol/L Tris，8 mol/L尿素，2 mol/L硫脲；采用pH 4~7的IPG胶条；上样方式选择被动的水化上样。等电聚焦过程中使用预设的缓慢升压模式，充分聚焦后选用合适的电压模式进行SDS-PAGE电泳，然后采用改良硝酸银法进行染色，最终获得了满意的蛋白质组双向电泳图谱。

Establishment of two-dimensional electrophoresis separation system for proteomes of human kidney tubular epithelial cells

BACKGROUND: Two-dimensional electrophoresis (2-DE) separation technology is one of core technologies for proteomics studies. However, the resolution of 2-DE map is highly altered due to different experimental conditions. Therefore, a 2-DE map with high resolution can be obtained via the optimization of key experimental conditions for a definite proteome sample.OBJECTIVE: To establish an effective separation system of 2-DE for the proteome of HK-2 human renal tubular epithelial cells. METHODS: HK-2 human renal tubular epithelial cells were cultured routinely. The proteins were extracted from HK-2 cells after lysing, and separated by 2-DE on the standard experimental condition with an optimization for each important experimental parameter. 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. The gray densities and numbers of protein spots, resolution and contaminating strands in 2-DE maps were determined.RESULTS AND CONCLUSION: A 2-DE separation system for proteome of human renal tubular epithelial cells was successfully established. The optimized experimental conditions were selected finally as follows: The lysis buffer containing 1% TBP, 4% CHAPS, 0.2% Bio-Lyte, 40 mmol/L Tris, 8 mol/L urea, 2 mol/L thiourea was applied to extract proteins from cells, and the passive rehydration proposal of samples was used to help the separation of large molecular weight proteins on the pH 4-7 IPG gels. 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. Under the optimized conditions, 2-DE maps with high resolution have been reproducibly obtained.