A toolbox for the immunomagnetic purification of signaling organelles. (15th January 2019)

• Record Type: Journal Article
• Authors: Fritsch, Jürgen; Tchikov, Vladimir; Hennig, Lena; Lucius, Ralph; Schütze, Stefan
• Is Part Of: Traffic. Volume 20:Number 3(2019)
• Page Start: 246
• Page End: 258
• Publication Date: 2019-01-15
• Subjects: cell death -- cell fractionation -- cytokine receptors -- organelles -- purification -- signal transduction; Biological transport -- Periodicals; 571.6
• DOI: 10.1111/tra.12631
• Languages: English
• ISSNs: 1398-9219

Abstract

Abstract : 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. Abstract : We present a set of protocols for the affinity purification of signaling organelles which is based on magnetic labelling and enrichment of organelles in a matrix‐free, high‐gradient magnetic separation device. The protocols allow purification of morphologically and functionally intact receptosomes/endosomes, lysosomes, exosomes and plasma membranes from one pool of cells.