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Real-Time Deubiquitination Assays Using a Free Ubiquitin Sensor

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Deubiquitinases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2591))

Abstract

Deubiquitinating enzymes cleave ubiquitin (Ub) from its attachment to another Ub, other proteins, peptides, or non-peptide adducts. In all cases, substrate hydrolysis by DUBs releases free Ub or polyubiquitin (polyUb) chains. Whereas most quantitative DUB assays depend on fluorescently labeled artificial substrates, employing a sensor able to detect Ub release in real time makes it possible to monitor DUB activity using virtually any Ub conjugate as a substrate. The protocols here describe the preparation of Atto532-tUI, a high-affinity sensor for free Ub, and its use in real-time deubiquitination assays.

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Acknowledgments

This work was supported in part by National Institutes of Health Grants R01GM115997 and R21GM135818 to R.E. Cohen. Y.-S. Choi was supported by the South Dakota Biomedical Research Infrastructure Network (SD BRIN) through an IDeA award from NIH-NIGMS (grant no. P20GM103443). The Usp2cc assays were supported by WestCore at BHSU.

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Authors and Affiliations

  1. School of Natural Sciences, Black Hills State University, Spearfish, SD, USA

    Yun-Seok Choi

  2. Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, USA

    Robert E. Cohen

Authors
  1. Yun-Seok Choi
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  2. Robert E. Cohen
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Corresponding author

Correspondence to Robert E. Cohen .

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Editors and Affiliations

  1. Microbiology & Cell Sci, Univ of Florida, Gainesville, FL, USA

    Julie Maupin-Furlow

  2. Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL, USA

    Mariola J. Edelmann

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Choi, YS., Cohen, R.E. (2023). Real-Time Deubiquitination Assays Using a Free Ubiquitin Sensor. In: Maupin-Furlow, J., Edelmann, M.J. (eds) Deubiquitinases. Methods in Molecular Biology, vol 2591. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2803-4_15

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  • DOI: https://doi.org/10.1007/978-1-0716-2803-4_15

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2802-7

  • Online ISBN: 978-1-0716-2803-4

  • eBook Packages: Springer Protocols

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