Assays of the affinity of a deubiquitinating enzyme for substrate, either through binding studies or determination of the Michaelis constant, KM, can shed light on substrate selectivity and the effects of mutations on substrate interactions. The difficulty in generating sufficient quantities of ubiquitinated substrate frequently presents a barrier to these studies. We describe here an alternative approach that can be used in cases where non-hydrolyzable, chemically ubiquitinated substrate analogs can be more readily generated. The substrate analog can be utilized as a competitive inhibitor in kinetics experiments monitoring cleavage of ubiquitin-AMC (Ub-AMC) by the deubiquitinating enzyme. The resulting inhibitory constant, Ki, provides a reliable approximation of the Kd for ubiquitinated substrate. We show how this approach can be used to assay the affinity of the yeast SAGA DUB module for nucleosomes containing monoubiquitinated H2B.
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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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