Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell LysatesPatrícia M. S. dos Passos *1, Valentine Spagnol *2, Camila R.S.T.B de Correia 1, Felipe R. Teixeira 1,2
1Department of Genetic and Evolution, Federal University of São Carlos, 2Department of Biochemistry and Immunology, Faculty of Medicine of Ribeirão Preto, University of São Paulo
We provide a detailed protocol for a ubiquitylation assay of a specific substrate and an E3 ubiquitin-ligase in mammalian cells. HEK293T cell lines were used for protein overexpression, the polyubiquitylated substrate was purified from cell lysates by immunoprecipitation, and resolved in SDS-PAGE. Immunoblotting was used to visualize this post-translational modification.
