Affinity Purification of the RNA Degradation Complex, the Exosome, from HEK-293 Cells

Michal Domanski; John LaCava

doi:10.21769/BioProtoc.2238

Affinity Purification of the RNA Degradation Complex, the Exosome, from HEK-293 Cells

Bio Protoc. 2017 Apr 20;7(8):e2238. doi: 10.21769/BioProtoc.2238.

Authors

Michal Domanski¹, John LaCava^{2

3}

Affiliations

¹ Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland.
² Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, USA.
³ Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, USA.

Abstract

The RNA exosome complex plays a central role in RNA processing and regulated turnover. Present both in cytoplasm and nucleus, the exosome functions through associations with ribonucleases and various adapter proteins (reviewed in [Kilchert et al., 2016]). The following protocol describes an approach to purify RNA exosome complexes from HEK-293 cells, making use of inducible ectopic expression, affinity capture, and rate-zonal centrifugation. The obtained RNA exosomes have been used successfully for proteomic, structural, and enzymatic studies (Domanski et al., 2016).

Keywords: Affinity capture; Cryomilling; EXOSC10; HEK-293 suspension culture; RNA exosome; Rate-zonal centrifugation.

Abstract

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