Protocol for pull-down capture of ubiquitinated and NEDDylated liver proteins from transgenic biotinylated ubiquitin and NEDD8 mice
Year: 2025
Type: article
Abstract: Ubiquitination and NEDDylation post-translational modifications (PTMs) are important regulatory mechanisms in liver disease. Here, we describe a protocol for capturing ubiquitinated and NEDDylated proteins by pull-down assay in liver samples from transgenic mouse models of whole-body biotinylated ubiquitin/NEDD8. We describe steps for sample preparation samples, protein immobilization, pull-down assay, and sample final elution. The identification of the hepatic ubiquitome and NEDDylome allows a better understanding of the pathophysiological mechanisms underlying liver disease. For complete details on the use and execution of this protocol, please refer to Gil-Pitarch et al. 1 • Preparation of hepatic proteins from biotinylated ubiquitin (Ub) and NEDD8 mice • Immobilization of biotinylated hepatic proteins to streptavidin-enriched agarose beads • Pull-down for capture of hepatic biotinylated ubiquitinated and NEDDylated proteins Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Ubiquitination and NEDDylation post-translational modifications (PTMs) are important regulatory mechanisms in liver disease. Here, we describe a protocol for capturing ubiquitinated and NEDDylated proteins by pull-down assay in liver samples from transgenic mouse models of whole-body biotinylated ubiquitin/NEDD8. We describe steps for sample preparation samples, protein immobilization, pull-down assay, and sample final elution. The identification of the hepatic ubiquitome and NEDDylome allows a better understanding of the pathophysiological mechanisms underlying liver disease.
Source: STAR Protocols
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $1,550
Funders: Gilead Sciences, “la Caixa” Foundation, Ministerio de Ciencia, Innovación y Universidades, Fundación Científica Asociación Española Contra el Cáncer, Federación Española de Enfermedades Raras, Ministerio de Ciencia e Innovación, Osasun Saila, Eusko Jaurlaritzako, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Instituto de Salud Carlos III, Spanish National Plan for Scientific and Technical Research and Innovation
OpenAlex ID: W4408728624