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目的:建立人参皂甙Rbl作用后大鼠神经细胞蛋白质组双向电泳分离体系,提高其分辨率和重复性。方法:人参皂甙Rbl 50mg/L作用于大鼠神经细胞,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了优化。结果:最终选择的裂解液配方是1%TBP,4%CHAPS,0.2%Bio-Lyte,40mmol/L Tris,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:成功建立了人参皂甙Rbl作用后大鼠神经细胞蛋白质组双向电泳分离体系,具有较高的分辨率和重复性。
OBJECTIVE: To establish a two dimensional gel electrophoresis separation system of rat neuronal cells after the action of ginsenoside Rb1, and to improve its resolution and repeatability. Methods: Ginsenoside Rbl 50mg / L was applied to nerve cells of rats and the whole protein was extracted. Proteins were separated by two-dimensional electrophoresis under standard conditions and the key factors were optimized. Results: The final lysate formulation was 1% TBP, 4% CHAPS, 0.2% Bio-Lyte, 40 mmol / L Tris, 8 mol / L urea and 2 mol / L thiourea. IPG strips with pH 4-7 were used for passive Hydration sample, isoelectric focusing using a slow boost mode, electrophoresis parameters adjusted according to Bio-Rad’s default program; modified silver nitrate method for protein spot staining. Thus obtaining a satisfactory proteome two-dimensional electrophoresis map. Conclusion: Two-dimensional gel electrophoresis separation system of rat neuronal cell has been successfully established after the action of ginsenoside Rbl, which has high resolution and repeatability.