Author:
Date:
2014-09-05
[Abstract] One way to study the function of plant mitochondria is to extract them from plant tissues in an uncontaminated, intact and functional form. The reductionist assumption is that the components present in such a preparation and the in vitro measurable functions or activities reliably reflect the in vivo properties of the organelle inside the plant cell. Here, we describe a method to isolate mitochondria from a relatively homogeneous plant tissue, the dormant potato tuber. The homogenization is done using a juice extractor, which is a relatively gentle homogenization procedure where the mitochondria are only exposed to strong shearing forces once. After removal of starch and large tissue pieces by filtration, differential centrifugation is used to remove residual starch as ...
[摘要] 研究植物线粒体功能的一种方法是以未污染,完整和功能形式从植物组织中提取植物线粒体。还原性假设是这种制剂中存在的组分和体外可测量的功能或活性可靠地反映植物细胞内的细胞器的体内性质。在这里,我们描述了从相对均匀的植物组织,休眠马铃薯块茎分离线粒体的方法。使用榨汁机进行均质化,其是相对温和的均质化程序,其中线粒体仅暴露于强剪切力一次。在通过过滤除去淀粉和大的组织碎片后,使用差速离心以除去残余淀粉以及更大的细胞器。然后首先通过使用步骤Percoll梯度来纯化粗线粒体。通过使用连续的Percoll梯度进一步纯化来自阶梯梯度的线粒体条带。梯度去除污染的淀粉体和过氧化物酶体以及破裂的线粒体。结果是高度纯化的,完整和功能的线粒体制剂,其可以在5%(v/v)二甲基亚砜存在下在液氮中冷冻并保存,以保持数月的完整性和功能性。
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