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Updated: May 28, 2026

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry
Published on: March 23, 2020
Analysis of protein ubiquitination
Jeffrey D Laney1, Mark Hochstrasser2
1Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island.
Abstract:
Attachment of ubiquitin (Ub) to a protein requires a series of enzymes that recognize the substrate and promote Ub transfer. Several methods are described in this unit for determining if a protein has Ub-transferring activity. They include immunoblotting of immunoprecipitated proteins, affinity purification using His-tagged Ub, assaying for auto-ubiquitination of E3, and assaying ubiquitination of a model substrate protein in vitro and in E. coli cells that express Ub-ligation enzymes. These methods are suitable for a variety of eukaryotic cells, but techniques are specifically described for use with yeast and mammalian cells.
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