Real-Time Deubiquitination Assays Using a Free Ubiquitin Sensor

Methods Mol Biol. 2023:2591:255-267. doi: 10.1007/978-1-0716-2803-4_15.

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.

Keywords: DUB assay; Deubiquitinase; Deubiquitination; Fluorescent ubiquitin sensor; Ubiquitin; tUI.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

MeSH terms

  • Hydrolysis
  • Polyubiquitin* / metabolism
  • Ubiquitin* / metabolism
  • Ubiquitination

Substances

  • Ubiquitin
  • Polyubiquitin