二次蔗糖密度梯度离心纯化线粒体

Mitochondrial Purification by Two Times Sucrose Density-gradient Centrifugation

该研究以小鼠肝组织和脑组织为原材料,对常规的差速离心法结合密度梯度离心纯化线粒体的方法进行改进,发现在常规操作步骤上增加一次非线性蔗糖密度梯度离心可以有效地提高线粒体纯度。透射电镜及免疫印迹鉴定结果显示,增加一次密度梯度离心后,线粒体纯度大大提高,而对线粒体得率影响有限。虽然这样延长了实验时间,但线粒体膜电位及琥珀酸脱氢酶比活实验证实,在线粒体纯度获得提高的同时,其活力并没有受到明显影响。因此,建议在进行线粒体蛋白质组检测等对线粒体纯度有较高要求的实验时,可以增加一次或者二次非线性蔗糖密度梯度离心,在维持线粒体活力的同时,提高其纯度,有利于后续实验的开展。

Mitochondrial fraction produced by differential centrifugation is often contaminated by other organelles and cytosolic components, and its further applications are thus limited. Density-gradient centrifugation is the most used strategy to improve the mitochondrial purity. However, mitochondria fraction obtained by density-gradient centrifugation is still slightly contaminated, In this report, we found an additional discontinuous sucrose density-gradient centrifugation will significantly improve the mitochondrial purity. Transmission electron microscopy and Western blot results suggested mitochondrial fraction isolated by differential centrifugation contained large amount of other organelles and cytosolic components. The isolated mitochondria were still contaminated by other impurities after one time discontinuous sucrose density-gradient centrifugation, although its purity was significantly enhanced. Impressively, the mitochondrial purity was highly improved by the second time centrifugation, and the yield was only slightly affected. The results of mitochondrial membrane potential and succinate dehydrogenase activity assay indicated that the mitochondrial membrane integrity and function were kept well with prolonged experimental procedure. Thus we suggest one or two additional density-gradient centrifugations could easily improve the mitochondria purity to facilitate further assays.