Real-Time Deubiquitination Assays Using a Free Ubiquitin Sensor.

Methods Mol Biol

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

Published: November 2022

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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http://dx.doi.org/10.1007/978-1-0716-2803-4_15DOI Listing

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