Studies in Saccharomyces cerevisiae and Schizosaccharomyces pombe have enhanced our understanding of the regulation and functions of histone H2B monoubiquitination (H2Bub1), a key epigenetic marker with important roles in transcription and other processes. The detection of H2Bub1 in yeasts using immunoblotting has been greatly facilitated by the commercial availability of antibodies against yeast histone H2B and the cross-reactivity of an antibody raised against monoubiquitinated human H2BK120. These antibodies have obviated the need to express epitope-tagged histone H2B to detect H2Bub1 in yeasts. Here, we provide a step-by-step protocol and best practices for the quantification of H2Bub1 in yeast systems, from cell extract preparation to immunoblotting using the commercially available antibodies. We demonstrate that the commercial antibodies can effectively and accurately detect H2Bub1 in S. cerevisiae and S. pombe. Further, we show that the C-terminal epitope-tagging of histone H2B alters the steady-state levels of H2Bub1 in yeast systems. We report a sectioned blot probing approach combined with the serial dilution of protein lysates and the use of reversibly stained proteins as loading controls that together provide a cost-effective and sensitive method for the quantitative evaluation of H2Bub1 in yeast.
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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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| Evidence ID | Analyze ID | File | Description |
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