Ubiquitin-conjugating enzymes (E2s) play a central role in ubiquitylation. They function to bridge the first, nonspecific step of ubiquitin activation by E1 with the transfer of activated ubiquitin to substrates by substrate-specific E3s. While sharing a common core UBC domain, members of this family exhibit significant specificity in their physical and functional interactions with E3s. Among the families of E2s, members of the yeast Ubc4/5 family are particularly well conserved in higher metazoans. In humans, these are represented by the UbcH5 family. Members of this ubiquitously expressed family show a capacity to interact with a wide range of E3s from both HECT and RING finger families, making them particularly useful tools in the laboratory. Using the UbcH5 family as a prototype, this chapter describes methods for the expression, purification, and characterization of E2 enzymes in vitro and some of the basics for their use in experiments in cells.
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| Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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| Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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| Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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| Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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| Site | Modification | Modifier | Source | Reference |
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| Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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| Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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| Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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| Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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