摘要
背景:神经干细胞是当今研究热点,双向电泳是蛋白质组学研究的技术基础,有必要建立稳定的神经干细胞蛋白质组双向凝胶电泳技术体系。目的:通过对神经干细胞双向凝胶电泳几个关键步骤的分析,建立稳定、重复性好的神经干细胞双向凝胶电泳的分离方法。方法:采用无血清体外细胞培养方法,培养胚胎大鼠神经干细胞并进行鉴定。提取样品蛋白并裂解,被动水化,采用不同IEF 参数,使用 PDQuest8.0 软件对图谱进行分析和比较。观察不同等点聚焦参数下双向电泳图谱质量,蛋白质点的数目及重复性。结果与结论:不同 IEF 参数条件下的双向电泳图谱的质量有所不同,增加低电压的除盐步骤有助于去除电泳胶图上的横纹。在 80 000 Vh 聚焦总能量下,获得较理想的电泳图。通过选用适当的 IEF 参数,建立了适当的神经干细胞双向凝胶电泳方法。
BACKGROUND：Neural stem cells are the current hot topic, and two-dimensional electrophoresis is the basic technique for proteome, it is necessary to establish a stable two-dimensional gel electrophoresis technology system for neural stem cells.OBJECTIVE：To establish a stable, reproducible two-dimensional gel electrophoresis separation method for neural stem cells through analysis of several key steps of two-dimensional electrophoresis.METHODS：Embryonic rat neural stem cells were cultured in serum-free cell culture.Proteins were extracted from samples and lysed.After passive rehydration, samples were isolated by different isoelectric focusing （IEF） parameters.Protein maps were analyzed by PDQuest8.0.Quality of two-dimensional gel electrophoresis maps under different IEF parameters, number and reproducibility of protein spots were detected.RESULTS AND CONCLUSION：Quality of two-dimensional gel electrophoresis diagrams differs with IEF parameters.Low-voltage step could remove the cross striations.Ideal electrophoresis could be obtained under 80 000 Vh.Proper two-dimensional gel electrophoresis methods can be constructed by optimizing IEF parameters.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2010年第36期6707-6710,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金资助项目(30771797)
项目名称:叶酸对神经干细胞增殖分化作用的蛋白质组学的研究
天津医科大学科学基金(2008Ky12)
项目名称:同型半胱氨酸对大鼠学习记忆及ERK信号通路的影响~~
