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.
Detection of ubiquitinated targets in mammalian and Drosophila models / E. Maspero, V. Fajner, J. Weber, S. Polo (METHODS IN ENZYMOLOGY). - In: Ubiquitin-dependent Protein Degradation / [a cura di] M. Hochstrasser. - [s.l] : Academic Press Inc., 2019. - ISBN 9780128186671. - pp. 293-318
Titolo: | Detection of ubiquitinated targets in mammalian and Drosophila models |
Autori: | POLO, SIMONA LAURA ANNA (Ultimo) (Corresponding) |
Parole Chiave: | Drosophila assay; ELISA-based assay; In vitro ubiquitination assay; Linkage-specific polyubiquitin antibodies; Substrate ubiquitination; TR-UB-FRET assay; Ubiquitin; Biochemistry; Molecular Biology |
Settore Scientifico Disciplinare: | Settore MED/04 - Patologia Generale |
Data di pubblicazione: | 2019 |
Digital Object Identifier (DOI): | http://dx.doi.org/10.1016/bs.mie.2018.12.030 |
Tipologia: | Book Part (author) |
Appare nelle tipologie: | 03 - Contributo in volume |
File in questo prodotto:
File | Descrizione | Tipologia | Licenza | |
---|---|---|---|---|
Maspero-chapter.pdf | Book Chapter | Publisher's version/PDF | Administrator Richiedi una copia |