Detection of ubiquitinated targets in mammalian and Drosophila models

Abstract

In this chapter, we describe techniques to detect ubiquitination events occurring in vivo. We focus on methodologies capable of preserving and detecting target protein ubiquitination in physiological conditions, without overexpressing a tagged version of ubiquitin. We provide detailed protocols for mammalian and Drosophila melanogaster systems using linkage-specific antibodies against ubiquitin. We analyze immunoblotting and immunofluorescence approaches as well as an ELISA-based quantitative approach, which allows comparing more samples and conditions. Finally, we describe the use of in vitro ubiquitination assays with purified proteins that allows a direct analysis of the molecular mechanisms behind specific E3 activity.

Keywords: Drosophila assay; ELISA-based assay; In vitro ubiquitination assay; Linkage-specific polyubiquitin antibodies; Substrate ubiquitination; TR-UB-FRET assay; Ubiquitin.