Detection of protein SUMOylation in vivo

Michael H. Tatham, Manuel S. Rodriguez, Dimitris P. Xirodimas, Ronald T. Hay

    Research output: Contribution to journalArticle

    104 Citations (Scopus)

    Abstract

    The small ubiquitin-like modifiers (SUMOs) are posttranslationally conjugated to eukaryotic cellular proteins with generally unpredictable consequences. SUMO substrates are found in many cellular systems, and functional analysis has revealed that substrate SUMOylation often has an important role in their regulation. Here we describe a cell-based protocol which can be used to detect the SUMOylation of a protein that relies on the enrichment of SUMO conjugates by purification of 6His-SUMO under denaturing conditions, followed by western blotting for the protein of interest. By purifying under denaturing conditions this method not only reduces the risk of false-positive identifications by non-covalent interactions, but also preserves SUMO-substrate conjugates by inhibiting SUMO-specific proteases-two caveats that may complicate other less stringent purification methods. In preliminary form, this protocol takes 4-5 d to perform, and it can be further elaborated to provide a multi-angled approach to investigate protein conjugation by SUMO.

    Original languageEnglish
    Pages (from-to)1363-1371
    Number of pages9
    JournalNature Protocols
    Volume4
    Issue number9
    DOIs
    Publication statusPublished - 2009

    Keywords

    • UBIQUITIN-LIKE PROTEINS
    • SUMO-2 TARGET PROTEINS
    • NUCLEAR-PORE COMPLEX
    • CONJUGATION
    • DEGRADATION
    • PROTEOMICS
    • MODIFIERS
    • STRATEGY
    • RANGAP1
    • UBC9

    Cite this