Identification and analysis of endogenous SUMO1 and SUMO2/3 targets in mammalian cells and tissues using monoclonal antibodies

• Verfasst von: Barysch, Sina-Victoria [VerfasserIn]
• Verfasst von: Dittner, Claudia [VerfasserIn]
• Verfasst von: Flotho, Annette [VerfasserIn]
• Verfasst von: Becker, Janina [VerfasserIn]
• Verfasst von: Melchior, Frauke [VerfasserIn]
• Titel: Identification and analysis of endogenous SUMO1 and SUMO2/3 targets in mammalian cells and tissues using monoclonal antibodies
• Verf.angabe: Sina V. Barysch, Claudia Dittner, Annette Flotho, Janina Becker, Frauke Melchior
• E-Jahr: 2014
• Jahr: 20 March 2014
• Umfang: 14 S.
• Fussnoten: Gesehen am 02.09.2020
• Titel Quelle: Enthalten in: Nature protocols
• Ort Quelle: Basingstoke : Nature Publishing Group, 2006
• Jahr Quelle: 2014
• Band/Heft Quelle: 9(2014), 4, Seite 896-909
• ISSN Quelle: 1750-2799
• DOI: doi:10.1038/nprot.2014.053
• Datenträger: Online-Ressource
• Sprache: eng
• K10plus-PPN: 1728632196

Abstract

SUMOylation is a protein modification that regulates the function of hundreds of proteins. Detecting endogenous SUMOylation is challenging: most small ubiquitin-related modifier (SUMO) targets are low in abundance, and only a fraction of a protein's cellular pool is typically SUMOylated. Here we present a step-by-step protocol for the enrichment of endogenous SUMO targets from mammalian cells and tissues (specifically, mouse liver), based on the use of monoclonal antibodies that are available to the scientific community. The protocol comprises (i) production of antibodies and affinity matrix, (ii) denaturing cell lysis, and (iii) SUMO immunoprecipitation followed by peptide elution. Production of affinity matrix and cell lysis requires ∼1 d. The immunoprecipitation with peptide elution can be performed in 2 d. As SUMO proteins are conserved, this protocol should also be applicable to other organisms, including many vertebrates and Drosophila melanogaster.