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Company: USA Scientific
Catalog#: 1111-3200
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NP-40 Fractionation and Nucleic Acid Extraction in Mammalian Cells
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2017-10-20
[Abstract]  This technique allows for efficient, highly purified cytoplasmic and nuclear-associated compartment fractionation utilizing NP-40 detergent in mammalian cells. The nuclear membrane is not disturbed during the fractionation thus leaving all nuclear and perinuclear associated components in the nuclear fraction. This protocol has been modified from Sambrook and Russell (2001) in order to downscale the amount of cells needed. To determine the efficiency of fractionation, we recommend using qPCR to compare the subcellular compartments that have been purified with equivalent amount of control whole cell extracts. [摘要]  该技术允许在哺乳动物细胞中利用NP-40洗涤剂进行高效,高度纯化的细胞质和核相关的分室分离。 在分离过程中核膜不受干扰,从而使核部分中的所有核和核周相关成分留下。 该协议已经从Sambrook和Russell(2001)修改,以便缩减所需的细胞数量。 为了确定分馏的效率,我们建议使用qPCR来比较已经用等量的对照全细胞提取物纯化的亚细胞室。
【背景】为了充分获得对细胞过程的理解,需要分离核和细胞质隔室。 有许多协议,甚至一些商业套件可用于帮助分离两个隔间。 然而,大多数需要高离心速度,产量差异甚至验证最终产品中的污染物量的方法也是很高的。 我们的协议在低速下使用小型台式离心机,以获得高纯度的细胞质提取物和核/核周组合相关隔室,以及数据分析,以验证污染物的百分比。 迄今为止,已经测试的细胞系是293T,HeLa和GHOST细胞系。 (Galvis,2014; Galvis等,,2014)。

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