A toolbox for the immunomagnetic purification of signaling organelles
A toolbox for the immunomagnetic purification of signaling organelles
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DOI：
10.1111/tra.12631
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发表时间：
2019-03-01
期刊：
影响因子：
4.5
通讯作者：
Schuetze, Stefan
中科院分区：
文献类型：
--
作者：
Fritsch, Juergen;Tchikov, Vladimir;Schuetze, Stefan
稳态和生物体和细胞的复杂功能依赖于在不同的细胞内和细胞外隔室中以及通过不同的介质进行的信号传导的复杂的空间和时间调节。我们在这里提出了一套快速和易于使用的方案，用于靶向特异性的免疫磁性富集的受体含有内体（受体体），质膜，溶酶体和外泌体。亚细胞器和外来体的分离是其后续详细的生化和功能分析的先决条件，并将推进其后续详细的生化和功能分析。不同子方案的顺序应用允许从一个细胞池中分离形态和功能完整的细胞器。富集基于使用受体配体或抗体与超顺磁性微珠一起进行选择性标记，然后在获得专利的无基质高梯度磁性纯化装置中分离。这种独特的磁室基于空分离柱外部的聚焦系统，产生聚焦在柱壁的高达3 T的高梯度磁场。
Homeostasis and the complex functions of organisms and cells rely on the sophisticated spatial and temporal regulation of signaling in different intra- and extracellular compartments and via different mediators. We here present a set of fast and easy to use protocols for the target-specific immunomagnetic enrichment of receptor containing endosomes (receptosomes), plasma membranes, lysosomes and exosomes. Isolation of subcellular organelles and exosomes is prerequisite for and will advance their detailed subsequent biochemical and functional analysis. Sequential application of the different subprotocols allows isolation of morphological and functional intact organelles from one pool of cells. The enrichment is based on a selective labelling using receptor ligands or antibodies together with superparamagnetic microbeads followed by separation in a patented matrix-free high-gradient magnetic purification device. This unique magnetic chamber is based on a focusing system outside of the empty separation column, generating an up to 3 T high-gradient magnetic field focused at the wall of the column.