Analysis of Protein Sumoylation

Kevin D. Sarge1

1 Department of Biochemistry, University of Kentucky, Lexington, Kentucky
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 14.8
DOI:  10.1002/0471140864.ps1408s83
Online Posting Date:  February, 2016
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Abstract

Sumoylation, wherein small ubiquitin‐like modifier (SUMO) proteins are covalently attached to specific lysine residues of target proteins, plays an important role in regulating many diverse cellular processes via its control of the functional properties of the modified proteins. Identification of new sumoylated proteins is expected to expand understanding of the role this modification has in cell function. This unit describes two different assays for determining whether a particular protein is sumoylated: the first method employs immunoprecipitation of the protein followed by SUMO immunoblot. The second involves incubating the protein (either an in vitro translation product or a purified recombinant protein) with a reconstituted in vitro sumoylation reaction followed by examination for increased molecular‐weight bands in SDS‐PAGE as sumoylated forms of the protein. Either of these approaches can also be used to determine the sumoylated lysine residue(s) by comparing modification of the normal protein versus lysine‐to‐arginine substitutions of potential sumoylation sites, which once determined allows analysis of the effect of sumoylation on the protein's function. © 2016 by John Wiley & Sons, Inc.

Keywords: SUMO‐1; SUMO‐2; SUMO‐3; sumoylation; post‐translational modification; Ubc9

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Immunoprecipitation of Target Proteins Followed by Anti‐SUMO Immunoblotting
  • Basic Protocol 2: In Vitro Sumoylation of a Candidate Protein Substrate Using a Reconstituted Enzymatic Reaction
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Immunoprecipitation of Target Proteins Followed by Anti‐SUMO Immunoblotting

  Materials
  • 100‐mm plates with cells expressing the protein of interest
  • Phosphate‐buffered saline (PBS; appendix 2E)
  • SDS lysis solution (see recipe)
  • PBS ( appendix 2E) with 0.5% (v/v) nonylphenyl‐polyethylene glycol (NP‐40; Sigma‐Aldrich) and 1× complete protease inhibitor (Roche)
  • N‐ethylmaleimide (optional)
  • Antibodies to the protein of interest or to the epitope tag of a transfected, tagged protein
  • IgG, species‐matched, nonspecific control (Sigma‐Aldrich)
  • Protein G–Sepharose or protein A–Sepharose (GE Healthcare)
  • 2× SDS‐PAGE sample buffer (see recipe)
  • Blocking buffer: e.g., 5% (w/v) nonfat dry milk
  • Antibodies to SUMO‐1, SUMO‐2, or SUMO‐3 (Thermo Fisher)
  • Enhanced chemiluminescence (ECL) or enhanced chemifluorescence (ECF) reagents (Thermo Fisher)
  • Plate scraper
  • 2‐ml microcentrifuge tubes
  • Refrigerated centrifuge
  • Sonicator
  • Dounce homogenizer (optional)
  • Micropipettor
  • End‐over‐end rotator
  • Additional reagents and equipment for SDS‐PAGE electrophoresis (Gallagher, ), electroblotting (Ursitti et al., ), and immunoblot detection (Gallagher, )

Basic Protocol 2: In Vitro Sumoylation of a Candidate Protein Substrate Using a Reconstituted Enzymatic Reaction

  Materials
  • In vitro‐coupled transcription/translation kit (e.g., Promega TNT T7 Quick for PCR DNA kit)
  • PCR product or plasmid containing an open reading frame (ORF) for the protein of interest
  • 10× sumoylation buffer (see recipe)
  • Creatine phosphokinase (Sigma‐Aldrich)
  • Creatine phosphate (Sigma‐Aldrich)
  • Inorganic pyrophosphatase (Sigma‐Aldrich)
  • SAE1/SAE2 heterodimer, purified (E1; LAE International, http://www.laebio.com)
  • Ubc9, purified (E2; IAE International, http://www.laebio.com)
  • SUMO‐1, SUMO‐2, or SUMO‐3 (purified; LAE International, http://www.laebio.com)
  • 2× SDS‐PAGE sample buffer (see recipe)
  • SDS‐PAGE fixing solution (see recipe)
  • Whatman filter paper
  • Additional reagents and equipment for SDS‐PAGE electrophoresis (Gallagher, ), electroblotting (Ursitti et al., ), and autoradiography (Gallagher, )
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Figures

Videos

Literature Cited

Literature Cited
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