Identification and Analysis of Endogenous SUMO1 and SUMO2/3 Targets in Mammalian Cells and Tissues Using Monoclonal Antibodies

Nat Protoc. 2014 Apr;9(4):896-909. doi: 10.1038/nprot.2014.053. Epub 2014 Mar 20.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal*
  • Humans
  • Immunoprecipitation / methods*
  • Liver / metabolism
  • Mice
  • SUMO-1 Protein / metabolism
  • Small Ubiquitin-Related Modifier Proteins / metabolism
  • Sumoylation*
  • Ubiquitins / metabolism

Substances

  • Antibodies, Monoclonal
  • SUMO-1 Protein
  • SUMO1 protein, human
  • SUMO2 protein, mouse
  • Small Ubiquitin-Related Modifier Proteins
  • Sumo3 protein, mouse
  • Ubiquitins