As part of collaborative research between The Netherlands Cancer Institute (NKI-AVL), Leiden University Medical Centre, UbiQ and the University of Amsterdam, van Kruijsbergen et al. report in Frontiers in Chemistry (2020, 8, article 111) detailed protocols for the development of antibodies recognizing site-specific ubiquitin modifications. Considerations for antigen design (please check out our UbiQ Block technology), screening and quality control are outlined.

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 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.

Figure 1. Outline of site-specific Ub antibody development. (i) design and synthesis of non-hydrolyzable Ub-peptide conjugates for immunization; (ii) design and synthesis of extended native iso-peptide linked Ub-peptide conjugates for screening; (iii) immunization, and generation and selection of hybridomas; (iv) selection of clones and antibody validation.

1. 1. 1. 1. 1. 1. 1. 1. 1. Division of Gene Regulation, Netherlands Cancer Institute, Amsterdam, Netherlands
                        2. Leiden Inst Chem Immunology, Oncode Institute, Leiden University Medical Center, Leiden, Netherlands
                        3. Division of Biochemistry, Netherlands Cancer Institute, Amsterdam, Netherlands
                        4. Division of Tumor Biology & Immunology, Netherlands Cancer Institute, Amsterdam, Netherlands
                        5. UbiQ Bio BV, Amsterdam, Netherlands
                        6. Department of Medical Biology, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands