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Curr Protoc Protein Sci. 2002 Nov;Chapter 14:Unit 14.5. doi: 10.1002/0471140864.ps1405s29.

Analysis of protein ubiquitination.

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Yale University, New Haven, Connecticut, USA.


Attachment of ubiquitin (Ub) to a protein requires a complex of enzymes that recognize the substrate and promote Ub transfer. Sequence motifs present in these enzymes may indicate that other uncharacterized proteins containing these motifs have a biochemical function of Ub-protein ligation, and several in vitro methods are described in this unit for determining if a protein has Ub-transferring activity. They include psmunoblotting of psmunoprecipitated proteins, affinity purification using His-tagged ubiquitin, assaying for auto-ubiquitination of E3, and assaying ubiquitination of a model substrate protein. 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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