Quantitative analysis of USP activity in vitro

Shreya Dharadhar, Robbert Q. Kim, Michael Uckelmann, Titia K. Sixma

Research output: Chapter in Book/Report/Conference proceedingChapter (Book)Otherpeer-review

3 Citations (Scopus)


Ubiquitin-specific proteases (USPs) are an important class of deubiquitinating enzymes (DUBs) that carry out critical roles in cellular physiology and are regulated at multiple levels. Quantitative characterization of USP activity is crucial for mechanistic understanding of USP function and regulation. This requires kinetic analysis using in vitro activity assays on minimal and natural substrates with purified proteins. In this chapter we give advice for efficient design of USP constructs and their optimal expression, followed by a series of purification strategies. We then present protocols for studying USP activity quantitatively on minimal and more natural substrates, and we discuss how to include possible regulatory elements such as internal USP domains or external interacting proteins. Lastly, we examine different binding assays for studying USP interactions and discuss how these can be included in full kinetic analyses.
Original languageEnglish
Title of host publicationUbiquitin and Ubiquitin-like Protein Modifiers
EditorsMark Hochstrasser
Place of PublicationCambridge, MA
PublisherAcademic Press
Number of pages39
ISBN (Print)9780128163597
Publication statusPublished - 2019

Publication series

NameMethods in Enzymology
PublisherAcademic Press


  • deubiquitinating enzymes
  • ubiquitin
  • ubiquitin-specific protease
  • protein purification
  • protein-protein interaction
  • quantiative enzymatic activity studies

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