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Plant Signalling Networks pp 163–172Cite as

In Vitro Protein Ubiquitination Assay

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 876))

Abstract

Ubiquitination is one of the most important posttranslational modifications in all eukaryote organisms. Ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3) are the three key enzymes in this process. To detect the specificity between E2 and E3 or enzyme–substrate relationship between E3 and a substrate protein, ubiquitination activity needs to be determined. This protocol provides a convenient and efficient in vitro assay for DTT-sensitive thioester formation of E2s and Ring/U-box-type E3s, and E3-mediated substrate ubiquitination. E2/E3 specificities can also be investigated quickly by using this system. This method can be applied to ubiquitination assays of proteins from any eukaryotic organisms.

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Acknowledgments

This research was supported by Grant CNSF31030047/90717006 from the National Natural Science Foundation of China and 973 Program 2011CB915402, which is a grant from National Basic Research Program of China. Q.X. is supported by grants from the Chinese Academy of Science.

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Authors and Affiliations

  1. State Key Laboratory of Plant Genomics, National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

    Qingzhen Zhao, Lijing Liu & Qi Xie

Authors
  1. Qingzhen Zhao
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  2. Lijing Liu
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  3. Qi Xie
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Corresponding author

Correspondence to Qi Xie .

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Editors and Affiliations

  1. Dept. Plant Biology, Carnegie Institution, Panama Street 260, Stanford, 94305, California, USA

    Zhi-Yong Wang

  2. Dept. Botany & Plant Science, University of California, Riverside, University Avenue 900, Riverside, 92521, California, USA

    Zhenbiao Yang

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© 2011 Springer Science+Business Media, LLC

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Zhao, Q., Liu, L., Xie, Q. (2011). In Vitro Protein Ubiquitination Assay. In: Wang, ZY., Yang, Z. (eds) Plant Signalling Networks. Methods in Molecular Biology, vol 876. Humana Press. https://doi.org/10.1007/978-1-61779-809-2_13

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  • DOI: https://doi.org/10.1007/978-1-61779-809-2_13

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-808-5

  • Online ISBN: 978-1-61779-809-2

  • eBook Packages: Springer Protocols

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