TY - JOUR
T1 - Strategy for Development of Site-Specific Ubiquitin Antibodies
AU - van Kruijsbergen, Ila
AU - Mulder, Monique P C
AU - Uckelmann, Michael
AU - van Welsem, Tibor
AU - de Widt, John
AU - Spanjaard, Aldo
AU - Jacobs, Heinz
AU - El Oualid, Farid
AU - Ovaa, Huib
AU - van Leeuwen, Fred
N1 - Copyright © 2020 van Kruijsbergen, Mulder, Uckelmann, van Welsem, de Widt, Spanjaard, Jacobs, El Oualid, Ovaa and van Leeuwen.
PY - 2020
Y1 - 2020
N2 - 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.
AB - 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.
U2 - https://doi.org/10.3389/fchem.2020.00111
DO - https://doi.org/10.3389/fchem.2020.00111
M3 - Article
C2 - 32154221
SN - 2296-2646
VL - 8
SP - 111
JO - Frontiers in Chemistry
JF - Frontiers in Chemistry
ER -