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 TITLE=Strategy for Development of Site-Specific Ubiquitin Antibodies JOURNAL=Frontiers in Chemistry VOLUME=8 YEAR=2020 URL=https://www.frontiersin.org/journals/chemistry/articles/10.3389/fchem.2020.00111 DOI=10.3389/fchem.2020.00111 ISSN=2296-2646 ABSTRACT=

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.