Immunodepletion and immunopurification as approaches for CSN research

Amnon Golan, Ning Wei, Elah Pick

    Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review


    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.

    Original languageEnglish
    Title of host publicationMethods in Molecular Biology
    PublisherHumana Press Inc.
    Number of pages14
    StatePublished - 1 Sep 2016

    Publication series

    NameMethods in Molecular Biology
    ISSN (Print)1064-3745

    Bibliographical note

    Publisher Copyright:
    © Springer Science+Business Media New York 2016.


    • COP9 signalosome
    • Cullin-RING ubiquitin ligase
    • Immunodepletion
    • Immunopurification
    • Nedd8

    ASJC Scopus subject areas

    • Molecular Biology
    • Genetics


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