Protocol for the purification and analysis of nuclear UFMylated proteins

• Published in: STAR protocols Vol. 6; no. 1; p. 103634
• Main Authors: Panichnantakul, Pudchalaluck, Oeffinger, Marlene
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 21.03.2025; Elsevier
• Subjects: Blotting, Western; Cell Biology; Cell Line; Cell Nucleus - chemistry; Cell Nucleus - metabolism; Cell separation/fractionation; HEK293 Cells; Humans; Mass Spectrometry - methods; Nuclear Proteins - chemistry; Nuclear Proteins - isolation & purification; Nuclear Proteins - metabolism; Proteomics; Cell separation/fractionation; Proteomics; Cell Biology
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2025.103634

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 [Display omitted] •Cell fractionation to separate nuclear and cytoplasmic UFMylated proteins•Affinity purification of UFMylated proteins from lysed and denatured nuclei•Semi-quantitative mass spectrometry to identify nuclear UFMylated proteins Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.