Quantitative Assessment of Histone H2B Monoubiquitination in Yeast Using Immunoblotting
Studies in <i>Saccharomyces cerevisiae</i> and <i>Schizosaccharomyces pombe</i> 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 d...
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MDPI AG
2022-09-01
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| Series: | Methods and Protocols |
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| Online Access: | https://www.mdpi.com/2409-9279/5/5/74 |
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| author | Andrew M. Leng Kaitlin S. Radmall Prakash K. Shukla Mahesh B. Chandrasekharan |
| author_facet | Andrew M. Leng Kaitlin S. Radmall Prakash K. Shukla Mahesh B. Chandrasekharan |
| author_sort | Andrew M. Leng |
| collection | DOAJ |
| description | Studies in <i>Saccharomyces cerevisiae</i> and <i>Schizosaccharomyces pombe</i> 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 <i>S. cerevisiae</i> and <i>S. pombe</i>. 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. |
| first_indexed | 2024-03-09T19:41:23Z |
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| institution | Directory Open Access Journal |
| issn | 2409-9279 |
| language | English |
| last_indexed | 2024-03-09T19:41:23Z |
| publishDate | 2022-09-01 |
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| series | Methods and Protocols |
| spelling | doaj.art-68a7aa3fe7024ad4ba506d4ae27e33dc2023-11-24T01:38:25ZengMDPI AGMethods and Protocols2409-92792022-09-01557410.3390/mps5050074Quantitative Assessment of Histone H2B Monoubiquitination in Yeast Using ImmunoblottingAndrew M. Leng0Kaitlin S. Radmall1Prakash K. Shukla2Mahesh B. Chandrasekharan3Department of Radiation Oncology, University of Utah School of Medicine, Salt Lake City, UT 84112, USADepartment of Radiation Oncology, University of Utah School of Medicine, Salt Lake City, UT 84112, USADepartment of Radiation Oncology, University of Utah School of Medicine, Salt Lake City, UT 84112, USADepartment of Radiation Oncology, University of Utah School of Medicine, Salt Lake City, UT 84112, USAStudies in <i>Saccharomyces cerevisiae</i> and <i>Schizosaccharomyces pombe</i> 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 <i>S. cerevisiae</i> and <i>S. pombe</i>. 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.https://www.mdpi.com/2409-9279/5/5/74histone H2B monoubiquitinationhistoneshistone modificationepigeneticsWestern blotimmunoblot |
| spellingShingle | Andrew M. Leng Kaitlin S. Radmall Prakash K. Shukla Mahesh B. Chandrasekharan Quantitative Assessment of Histone H2B Monoubiquitination in Yeast Using Immunoblotting Methods and Protocols histone H2B monoubiquitination histones histone modification epigenetics Western blot immunoblot |
| title | Quantitative Assessment of Histone H2B Monoubiquitination in Yeast Using Immunoblotting |
| title_full | Quantitative Assessment of Histone H2B Monoubiquitination in Yeast Using Immunoblotting |
| title_fullStr | Quantitative Assessment of Histone H2B Monoubiquitination in Yeast Using Immunoblotting |
| title_full_unstemmed | Quantitative Assessment of Histone H2B Monoubiquitination in Yeast Using Immunoblotting |
| title_short | Quantitative Assessment of Histone H2B Monoubiquitination in Yeast Using Immunoblotting |
| title_sort | quantitative assessment of histone h2b monoubiquitination in yeast using immunoblotting |
| topic | histone H2B monoubiquitination histones histone modification epigenetics Western blot immunoblot |
| url | https://www.mdpi.com/2409-9279/5/5/74 |
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