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  3. Protocol for the purification and analysis of nuclear UFMylated proteins

Protocol for the purification and analysis of nuclear UFMylated proteins

  • STAR Protoc. 2025 Mar 21;6(1):103634. doi: 10.1016/j.xpro.2025.103634.
Pudchalaluck Panichnantakul 1,  Marlene Oeffinger 2
Affiliations

Affiliations

  • 1 Institut de Recherches Cliniques de Montréal, Center for Genetic and Neurological Diseases, 110 Avenue des Pins Ouest, Montréal, Québec H2W 1R7, Canada; Division of Experimental Medicine, Faculty of Medicine, McGill University, Montréal, Québec H4A 3J1, Canada.
  • 2 Institut de Recherches Cliniques de Montréal, Center for Genetic and Neurological Diseases, 110 Avenue des Pins Ouest, Montréal, Québec H2W 1R7, Canada; Division of Experimental Medicine, Faculty of Medicine, McGill University, Montréal, Québec H4A 3J1, Canada; Département de Biochimie et Médicine Moléculaire, Faculté de Médicine, Université de Montréal, Montréal, Québec H3C 3J7, Canada. Electronic address: marlene.oeffinger@ircm.qc.ca.
PMID: 39937649 DOI: 10.1016/j.xpro.2025.103634
Abstract

Protein UFMylation regulates numerous cellular processes including ribosome quality control and nuclear DNA repair. Here, we present a technique to isolate nuclei and purify UFMylated proteins under denaturing non-reducing conditions from commonly used mammalian cell line models such as hTERT-RPE1, HEK293, U2OS, and HCT116 cells. We then describe procedures for identifying and analyzing purified UFMylated proteins using mass spectrometry and western blot. For complete details on the use and execution of this protocol, please refer to Panichnantakul et al.1.

Keywords

Cell Biology; Cell separation/fractionation; Proteomics.

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