人急性早幼粒HL60细胞双向电泳条件的优化

Optimization for two-dimensional electrophoresis analysis of human acute premyelocyte HL60 cell line

目的:比较不同蛋白提取方式和不同电泳条件下HL60细胞全蛋白的双向电泳结果,寻找新的针对HL60细胞省时高效的双向电泳条件.方法:分别采用标准蛋白提取,三氯乙酸/丙酮沉淀和超声破碎三种不同蛋白提取方法,在传统双向电泳和改良双向电泳条件下做对比,并做蛋白点数和凝胶重复性分析.结果:在传统等电聚焦条件下,双向电泳凝胶图谱两端均出现大量横纹和阴影,分离效果较差.采用改良双向电泳条件后,三种蛋白提取方法的蛋白分离效果均明显改善,重复性提高,并且省时、可靠.其中,标准蛋白提取优于三氯乙酸/丙酮沉淀和超声破碎两种蛋白提取方法,蛋白点数即可达到(1102±7)个,重复性为(74.8±6.4)%,基本满足后续蛋白点的筛选和比对.三氯乙酸/丙酮沉淀法可造成部分蛋白损失,仅能分离出(844±15)个蛋白点.超声破碎法虽然使得蛋白点数明显增多,但可重复性明显下降,还不足40%.结论:采用标准蛋白提取法和改良的电泳条件,可以省时、高效地分离HL60细胞全蛋白.

AIM： To compare different methods of protein extraction and electrophoretic condition in two-dimensional electrophoresis analysis of human acute premyelocyte HL60 cell line, so as to find a method which is more efficient and rapid. METHODS： Three different methods of protein extraction, including typical protein extraction, precipitation of TCA/acetone and supersound quassation, were used respectively in the traditional electrophoresis condition and the improved electrophoresis conditions. The results of protein separation spots and electrophoretic repeatability were analyzed. RESULTS： There were lots of transverse striations and vague outlines on the amphi-SDSPAGE gels using different methods of protein extraction in the traditional electrophoretic condition. The improved electrophoretic condition not only significantly improved the protein spots＇ separation and repeatability, but also saved time. Using the typical protein extraction method and the new isoelectrofocusing method, the mimber of protein spots was increased to 1102 ± 7 and the result of protein spots＇ repeatability was （74.8 ± 6.4） %, which meet the requirement of the study. The method of TCA/acetone precipitation caused some loss of protein, and the number of separated protein spots was 844 ± 15 ; with the method of supersound quassation, the number of separated protein spots was increased, but repeatability was decreased significantly, being less than 40%. CONCLUSION： The protein spot separation can be significantly improved by the typical protein extraction method and in the improved electrophoretic condition.