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Proteostasis pp 103-116 | Cite as

Immunodepletion and Immunopurification as Approaches for CSN Research

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1449)

Abstract

The COP9 signalosome (CSN) is an evolutionary conserved complex that is found in all eukaryotes, and implicated in regulating the activity of Cullin-RING ubiquitin Ligases (CRLs). Activity of CRLs is highly regulated; complexes are active when the cullin subunit is covalently attached to the ubiquitin like modifier, Nedd8. Neddylation/deneddylation cycles are required for proper CRLs activity, and deneddylation is performed by the CSN complex.

We describe here a method utilizing resin-coupled antibodies to deplete the CSN from human cell extracts, and to obtain endogenous CSN complexes by immunopurification. In the first step, the cross-linked primary antibodies recognize endogenous CSN complexes, and deplete them from cell extract as the extract passes through the immunoaffinity column. The resulting “CSN-depleted extract” (CDP) is rich in neddylated cullins that can be used as a substrate for cullin-deneddylation assay for CSN complexes purified from various eukaryotes. Consequently, regeneration of the column results in dissociation of a highly purified CSN complex, together with its associated proteins. Immunopurification of the CSN from various human tissues or experimental conditions is advantageous for the generation of numerous CSN-interaction maps.

Key words

Nedd8 COP9 signalosome Cullin-RING ubiquitin ligase Immunodepletion Immunopurification 
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Notes

Acknowledgements

We would like to thank the Technion-Haifa Joint Research Grant Program for funding our studies; the laboratory of Prof. Yuval Shoham, at the Biotechnology and Food Engineering Faculty at the Technion, Israel, for professional support. The authors would like to acknowledge networking support by the Proteostasis COST Action (BM1307).

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Biology and EnvironmentUniversity of Haifa at OranimTivonIsrael
  2. 2.Protein Purification CenterRamat YohananIsrael
  3. 3.Department of Molecular, Cellular and Developmental BiologyYale UniversityNew HavenUSA

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