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Strategy for Development of Site-Specific Ubiquitin Antibodies.

Authors :
van Kruijsbergen I
Mulder MPC
Uckelmann M
van Welsem T
de Widt J
Spanjaard A
Jacobs H
El Oualid F
Ovaa H
van Leeuwen F
Source :
Frontiers in chemistry [Front Chem] 2020 Feb 21; Vol. 8, pp. 111. Date of Electronic Publication: 2020 Feb 21 (Print Publication: 2020).
Publication Year :
2020

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.<br /> (Copyright © 2020 van Kruijsbergen, Mulder, Uckelmann, van Welsem, de Widt, Spanjaard, Jacobs, El Oualid, Ovaa and van Leeuwen.)

Details

Language :
English
ISSN :
2296-2646
Volume :
8
Database :
MEDLINE
Journal :
Frontiers in chemistry
Publication Type :
Academic Journal
Accession number :
32154221
Full Text :
https://doi.org/10.3389/fchem.2020.00111