Detection of ubiquitinated targets in mammalian and Drosophila models.
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.
['Maspero|Elena|E|', 'Fajner|Valentina|V|', 'Weber|Janine|J|', 'Polo|Simona|S|']
D000818:Animals D029721:Drosophila Proteins D004331:Drosophila melanogaster D004797:Enzyme-Linked Immunosorbent Assay D005455:Fluorescent Antibody Technique D006367:HeLa Cells D006801:Humans D015151:Immunoblotting D025821:Polyubiquitin D025801:Ubiquitin D044767:Ubiquitin-Protein Ligases D054875:Ubiquitination
Ubiquitin In vitro ubiquitination assay Drosophila assay ELISA-based assay Linkage-specific polyubiquitin antibodies Substrate ubiquitination TR-UB-FRET assay