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Strategy for Development of Site-Specific Ubiquitin Antibodies

Protein ubiquitination is a key post-translational modification regulating a wide range of biological processes. Ubiquitination involves the covalent attachment of the small protein ubiquitin to a lysine of a protein substrate. In addition to its well-established role in protein degradation, protein...

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Autores principales: van Kruijsbergen, Ila, Mulder, Monique P. C., Uckelmann, Michael, van Welsem, Tibor, de Widt, John, Spanjaard, Aldo, Jacobs, Heinz, El Oualid, Farid, Ovaa, Huib, van Leeuwen, Fred
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7047734/
https://www.ncbi.nlm.nih.gov/pubmed/32154221
http://dx.doi.org/10.3389/fchem.2020.00111
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author van Kruijsbergen, Ila
Mulder, Monique P. C.
Uckelmann, Michael
van Welsem, Tibor
de Widt, John
Spanjaard, Aldo
Jacobs, Heinz
El Oualid, Farid
Ovaa, Huib
van Leeuwen, Fred
author_facet van Kruijsbergen, Ila
Mulder, Monique P. C.
Uckelmann, Michael
van Welsem, Tibor
de Widt, John
Spanjaard, Aldo
Jacobs, Heinz
El Oualid, Farid
Ovaa, Huib
van Leeuwen, Fred
author_sort van Kruijsbergen, Ila
collection PubMed
description Protein ubiquitination is a key post-translational modification regulating a wide range of biological processes. Ubiquitination involves the covalent attachment of the small protein ubiquitin to a lysine of a protein substrate. In addition to its well-established role in protein degradation, protein ubiquitination plays a role in protein-protein interactions, DNA repair, transcriptional regulation, and other cellular functions. Understanding the mechanisms and functional relevance of ubiquitin as a signaling system requires the generation of antibodies or alternative reagents that specifically detect ubiquitin in a site-specific manner. However, in contrast to other post-translational modifications such as acetylation, phosphorylation, and methylation, the instability and size of ubiquitin−76 amino acids–complicate the preparation of suitable antigens and the generation antibodies detecting such site-specific modifications. As a result, the field of ubiquitin research has limited access to specific antibodies. This severely hampers progress in understanding the regulation and function of site-specific ubiquitination in many areas of biology, specifically in epigenetics and cancer. Therefore, there is a high demand for antibodies recognizing site-specific ubiquitin modifications. Here we describe a strategy for the development of site-specific ubiquitin antibodies. Based on a recently developed antibody against site-specific ubiquitination of histone H2B, we provide detailed protocols for chemical synthesis methods for antigen preparation and discuss considerations for screening and quality control experiments.
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spelling pubmed-70477342020-03-09 Strategy for Development of Site-Specific Ubiquitin Antibodies van Kruijsbergen, Ila Mulder, Monique P. C. Uckelmann, Michael van Welsem, Tibor de Widt, John Spanjaard, Aldo Jacobs, Heinz El Oualid, Farid Ovaa, Huib van Leeuwen, Fred Front Chem Chemistry Protein ubiquitination is a key post-translational modification regulating a wide range of biological processes. Ubiquitination involves the covalent attachment of the small protein ubiquitin to a lysine of a protein substrate. In addition to its well-established role in protein degradation, protein ubiquitination plays a role in protein-protein interactions, DNA repair, transcriptional regulation, and other cellular functions. Understanding the mechanisms and functional relevance of ubiquitin as a signaling system requires the generation of antibodies or alternative reagents that specifically detect ubiquitin in a site-specific manner. However, in contrast to other post-translational modifications such as acetylation, phosphorylation, and methylation, the instability and size of ubiquitin−76 amino acids–complicate the preparation of suitable antigens and the generation antibodies detecting such site-specific modifications. As a result, the field of ubiquitin research has limited access to specific antibodies. This severely hampers progress in understanding the regulation and function of site-specific ubiquitination in many areas of biology, specifically in epigenetics and cancer. Therefore, there is a high demand for antibodies recognizing site-specific ubiquitin modifications. Here we describe a strategy for the development of site-specific ubiquitin antibodies. Based on a recently developed antibody against site-specific ubiquitination of histone H2B, we provide detailed protocols for chemical synthesis methods for antigen preparation and discuss considerations for screening and quality control experiments. Frontiers Media S.A. 2020-02-21 /pmc/articles/PMC7047734/ /pubmed/32154221 http://dx.doi.org/10.3389/fchem.2020.00111 Text en Copyright © 2020 van Kruijsbergen, Mulder, Uckelmann, van Welsem, de Widt, Spanjaard, Jacobs, El Oualid, Ovaa and van Leeuwen. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Chemistry
van Kruijsbergen, Ila
Mulder, Monique P. C.
Uckelmann, Michael
van Welsem, Tibor
de Widt, John
Spanjaard, Aldo
Jacobs, Heinz
El Oualid, Farid
Ovaa, Huib
van Leeuwen, Fred
Strategy for Development of Site-Specific Ubiquitin Antibodies
title Strategy for Development of Site-Specific Ubiquitin Antibodies
title_full Strategy for Development of Site-Specific Ubiquitin Antibodies
title_fullStr Strategy for Development of Site-Specific Ubiquitin Antibodies
title_full_unstemmed Strategy for Development of Site-Specific Ubiquitin Antibodies
title_short Strategy for Development of Site-Specific Ubiquitin Antibodies
title_sort strategy for development of site-specific ubiquitin antibodies
topic Chemistry
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7047734/
https://www.ncbi.nlm.nih.gov/pubmed/32154221
http://dx.doi.org/10.3389/fchem.2020.00111
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