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Updated: Jul 12, 2025
Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates
Published on: May 10, 2022
Preparation of site-specifically fluorophore-labeled polyubiquitin chains for FRET studies of Cdc48 substrate
Cameron Williams1, Ken C Dong2, Connor Arkinson2
1Biophysics Graduate Group, University of California, Berkeley, CA 94720, USA; California Institute for Quantitative Biosciences, University of California at Berkeley, Berkeley, CA 94720, USA.
Abstract:
A critical step in the removal of polyubiquitinated proteins from macromolecular complexes and membranes for subsequent proteasomal degradation is the unfolding of an ubiquitin moiety by the cofactor Ufd1/Npl4 (UN) and its insertion into the Cdc48 ATPase for mechanical translocation. Here, we present a stepwise protocol for the assembly and purification of Lys48-linked ubiquitin chains that are fluorophore labeled at specific ubiquitin moieties and allow monitoring polyubiquitin engagement by the Cdc48-UN complex in a FRET-based assay. For complete details on the use and execution of this protocol, please refer to Williams et al. (2023).1.
