ISOLATION OF THE MAJOR SUBCELLULAR ORGANELLES FROM MOUSE-LIVER USING NYCODENZ GRADIENTS WITHOUT THE USE OF AN ULTRACENTRIFUGE
ISOLATION OF THE MAJOR SUBCELLULAR ORGANELLES FROM MOUSE-LIVER USING NYCODENZ GRADIENTS WITHOUT THE USE OF AN ULTRACENTRIFUGE
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DOI：
10.1016/0003-2697(90)90463-j
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发表时间：
1990-06-01
影响因子：
2.9
通讯作者：
RICKWOOD, D
中科院分区：
文献类型：
--
作者：
GRAHAM, JM;FORD, T;RICKWOOD, D
通常，亚细胞细胞器如细胞核、线粒体、溶酶体和高尔基体膜首先通过在低速或高速离心机中的差速离心分离，然后通过在超速离心机中的梯度离心纯化。我们使用一种新的高速离心机（最大28，000 rpm）制备这些细胞器，该离心机可以产生比标准机器更高的径向离心力（rcfs）。我们已经表明，大多数亚细胞器可以通过使用低粘度Nycodenz梯度在rcfs低于通常使用的ultracentheges，而不增加离心时间进行纯化。使用Nycodenz还可以从梯度中快速收获材料，我们已经采用了许多酶测定法来促进梯度分析。
Commonly, subcellular organelles such as nuclei, mitochondria, lysosomes, and Golgi membranes are isolated first by differential centrifugation in low-speed or high-speed centrifuges and then purified by gradient centrifugation in ultracentrifuges. We have prepared these organelles using a new high-speed centrifuge (28,000 rpm max) which allows the generation of higher radial centrifugal forces (rcfs) than are available in standard machines. We have shown that most subcellular organelles can be purified by using low-viscosity Nycodenz gradients at rcfs lower than those normally used in ultracentrifuges, without increasing the time of centrifugation. Use of Nycodenz also allows rapid harvesting of material from gradients and we have adapted a number of enzyme assays to facilitate gradient analyses.