Real-Time Surface Plasmon Resonance (SPR) for the Analysis of Interactions Between SUMO Traps and Mono- or PolySUMO Moieties

Methods Mol Biol. 2016:1475:99-107. doi: 10.1007/978-1-4939-6358-4_7.

Abstract

Isolating endogenous SUMOylated proteins is a challenging task due to the high reversibility of this posttranslational modification. We have shown that SUMO traps are useful tools for the enrichment and isolation of proteins modified by SUMO in vitro and in vivo. To characterize the affinity and specificity of different SUMO chains for these traps, that are based on SUMO-interacting motifs, we have used real-time surface plasmon resonance (SPR), which allows a label-free analysis of protein/protein interactions. Here, a protocol to determine the affinities of multivalent SUMO traps for polySUMO chains or mono-SUMO molecules by SPR is presented.

Keywords: Affinity constants; Avidity; Multivalence; Protein interactions; SPR; SUMO-binding entities.

MeSH terms

  • Antibodies / chemistry
  • Cloning, Molecular
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Gene Expression
  • Genetic Vectors / chemistry
  • Genetic Vectors / metabolism
  • Glutathione Transferase / genetics
  • Glutathione Transferase / metabolism
  • Humans
  • Microchip Analytical Procedures
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Protein Processing, Post-Translational*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • SUMO-1 Protein / genetics
  • SUMO-1 Protein / metabolism*
  • Small Ubiquitin-Related Modifier Proteins / genetics
  • Small Ubiquitin-Related Modifier Proteins / metabolism*
  • Sumoylation
  • Surface Plasmon Resonance / methods*

Substances

  • Antibodies
  • Recombinant Fusion Proteins
  • SUMO-1 Protein
  • SUMO1 protein, human
  • SUMO2 protein, human
  • Small Ubiquitin-Related Modifier Proteins
  • Glutathione Transferase