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Isolation of ubiquitinated substrates by tandem affinity purification of E3 ligase–polyubiquitin-binding domain fusions (ligase traps)

Nature Protocols volume 11, pages 291–301 (2016)Cite this article

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Abstract

Ubiquitination is an essential protein modification that influences eukaryotic processes ranging from substrate degradation to nonproteolytic pathway alterations, including DNA repair and endocytosis. Previous attempts to analyze substrates via physical association with their respective ubiquitin ligases have had some success. However, because of the transient nature of enzyme-substrate interactions and rapid protein degradation, detection of substrates remains a challenge. Ligase trapping is an affinity purification approach in which ubiquitin ligases are fused to a polyubiquitin-binding domain, which allows the isolation of ubiquitinated substrates. Immunoprecipitation is first used to enrich for proteins that are bound to the ligase trap. Subsequently, affinity purification is used under denaturing conditions to capture proteins conjugated with hexahistidine-tagged ubiquitin. By using this protocol, ubiquitinated substrates that are specific for a given ligase can be isolated for mass spectrometry or western blot analysis. After cells have been collected, the described protocol can be completed in 2–3 d.

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Figure 1: Overview of the ligase trapping procedure.
Figure 2: Quality-control post-purification results for yeast purification.
Figure 3: Quality-control post-purification results for mammalian purification.

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Acknowledgements

This work was supported by US National Institutes of Health (NIH) grants R01 GM059691 and GM070539 to D.P.T., and by a predoctoral fellowship from the American Heart Association to T.B.L.

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  1. Kevin G Mark and Theresa B Loveless: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry and Biophysics, Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, California, USA

    Kevin G Mark, Theresa B Loveless & David P Toczyski

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  1. Kevin G Mark
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Contributions

K.G.M. developed the protocol for yeast application, designed and performed experiments, analyzed data and wrote the manuscript; T.B.L. developed the protocol for mammalian cell application, designed and performed experiments, analyzed data and wrote the manuscript; D.P.T. conceived and oversaw the study, designed experiments, analyzed data and wrote the manuscript.

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Correspondence to David P Toczyski.

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The authors declare no competing financial interests.

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Mark, K., Loveless, T. & Toczyski, D. Isolation of ubiquitinated substrates by tandem affinity purification of E3 ligase–polyubiquitin-binding domain fusions (ligase traps). Nat Protoc 11, 291–301 (2016). https://doi.org/10.1038/nprot.2016.008

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