【作者】 肖华； 和雨晨； 孔凡志； 樊柳荫； 曹成喜；

【Author】 Hua Xiao;Yuchen He;Zhifan Kong;Liu-Yin Fan;Cheng-Xi Cao;School of Life Sciences and Biotechnology, Shanghai Jiao Tong University;

【机构】 上海交通大学生命科学技术学院；

【摘要】 线粒体是重要的细胞器,其分离纯化极具挑战性。常用的密度梯度离心技术不能区分与线粒体类似的其它细胞器,因此有必要发展新装置来实现线粒体的富集。在前期自由流电泳工作的基础上,我们构建了循环式自由流等电聚焦(FFIEF)装置,采用两套泵实现电极液的输送和缓冲液的循环。为减少电泳缓冲液和电场对线粒体的损伤,我们发展了样品进样新模式,即在分离腔内形成p H梯度后再进样。我们从兔肝中先通过差速离心得到线粒体粗品,再进行自由流等电聚焦电泳分离制备线粒体组份。扫描电镜结果显示在p H6.24和6.61两个组份中的线粒体形态完整,统计分析显示线粒体纯度显著升高。随着组份p H值的降低,线粒体的结构逐渐残缺。线粒体膜电位的测定显示完整的线粒体和不完整的线粒体有显著差别。蛋白质组学分析结果表明,和残缺的线粒体和线粒体粗品相比,从完整的线粒体中可以多鉴定20%以上的蛋白。实验证明循环式自由流等电聚焦电泳可以用于线粒体的有效分离纯化。更多还原

【Abstract】 Mitochondria are important cell organelle, while their purification is challenging. Based on our previous work on free-flow electrophoresis, we build novel recycling free-flow isoelectric focusing system. Dual pumps were employed to transfer buffers. In order to minimize the hurt from running buffer and electric field, we developed post-p H gradient injection method. Crude mitochondria were extracted from rabbit liver through differential centrifugation. After FFIEF, TEM images demonstrated that the morphology of mitochondria at pH 6.21 and 6.61 are intact. Statistical analysis proved the high purity of mitochondria. The lower the pH, the worse their morphology. Membrane potential data showed that there was significant difference between intact and broken mitochondria. Proteomics analysis again demonstrated that 20% more proteins could be identified from intact ones than that of broken or crude mitochondria. Experimental results collectively proved that FFIEF is an efficient approach for mitochondria purification.更多还原