A protocol for the subcellular fractionation of Saccharomyces cerevisiae using nitrogen cavitation and density gradient centrifugation.
A protocol for the subcellular fractionation of Saccharomyces cerevisiae using nitrogen cavitation and density gradient centrifugation.
复制标题
使用氮空化和密度梯度离心对酿酒酵母进行亚细胞分级的方案。
DOI：
10.1002/yea.3002
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
Wang Y
中科院分区：
文献类型：
--
作者：
Wang Y
酵母亚细胞分离的大多数方案涉及使用机械剪切力来裂解酵母细胞壁酶解产生的球质体。这些机械均质程序通常涉及手动使用设备，如Dounce均质机，因此非常依赖于操作员，因此缺乏可重复性。在这里，我们报告了一种高度可重复的方法均质酵母细胞基于氮空化。这已被优化，以允许有效释放亚细胞区室，显示出高度的完整性。该方案在一系列样品体积和缓冲环境中保持有效和可重复性。随后的分离方法，采用蔗糖和碘二醇密度梯度，已经发展到分离s的主要膜结合区室。酵母。我们提出了一种快速、简便、稳健和高效的综合方案，这将使s的亚细胞区室的“组学”研究成为可能。酵母和其他酵母。©2014作者。《酵母》由约翰威利父子有限公司出版。
Most protocols for yeast subcellular fractionation involve the use of mechanical shear forces to lyse the spheroplasts produced by the enzymatic digestion of theSaccharomyces cerevisiaecell wall. These mechanical homogenization procedures often involve the manual use of devices such as the Dounce homogenizer, and so are very operator‐dependent and, in consequence, lack reproducibility. Here, we report a highly reproducible method of homogenizing yeast cells based on nitrogen cavitation. This has been optimized to allow efficient release of subcellular compartments that show a high degree of integrity. The protocol remains effective and reproducible across a range of sample volumes and buffer environments. The subsequent separation method, which employs both sucrose and iodixanol density gradients, has been developed to resolve the major membrane‐bound compartments ofS. cerevisiae. We present an integrated protocol that is fast, facile, robust and efficient and that will enable ‘omics’ studies of the subcellular compartments ofS. cerevisiaeand other yeasts. © 2014 The Authors. Yeast published by John Wiley & Sons Ltd.