Protein Profiling Using Two‐Dimensional Difference Gel Electrophoresis (2‐D DIGE)

Renata Feret1, Kathryn S. Lilley1

1 Department of Biochemistry, University of Cambridge, Cambridge
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 22.2
DOI:  10.1002/0471140864.ps2202s75
Online Posting Date:  February, 2014
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Abstract

2‐D DIGE relies on pre‐electrophoretic labeling of samples with one of three spectrally distinct fluorescent dyes, followed by electrophoresis of all samples in one 2‐D gel. The dye‐labeled samples are then viewed individually by scanning the gel at different wavelengths, which circumvents problems with gel‐to‐gel variation and spot matching between gels. Image analysis programs are used to generate volume ratios for each spot, which essentially describe the intensity of a particular spot in each test sample, and thus enable protein abundance level changes to be identified and quantified. This unit describes the 2‐D DIGE procedure including sample preparation from various cell types, labeling of proteins, and points to consider in the downstream processing of fluorescently labeled samples. Curr. Protoc. Protein Sci. 75:22.2.1‐22.2.17. © 2014 by John Wiley & Sons, Inc.

Keywords: DIGE; 2‐D gel electrophoresis; quantitative; proteomics

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Minimal Labeling of Protein for 2‐D DIGE (N‐Hydroxysuccinimide Ester Derivatized Cyanine Dyes that Label Primary Amines)
  • Support Protocol 1: Preparation of Protein for CY Labeling Reactions
  • Support Protocol 2: Protein Identification from DIGE Gels by Mass Spectrometric Techniques
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Minimal Labeling of Protein for 2‐D DIGE (N‐Hydroxysuccinimide Ester Derivatized Cyanine Dyes that Label Primary Amines)

  Materials
  • Protein sample (see protocol 2)
  • Protein determination kit (e.g., BioRad DC protein assay, RC DC protein assay, Quick Start Bradford protein assay from BioRad Laboratories; or 2‐D Quant kit from GE Healthcare Life Science)
  • Protein concentration kit (e.g., 2‐D Clean‐Up kit, GE Healthcare Life Science; or PerfectFOCUS, Genotech), optional
  • 0.1 M ammonium acetate in methanol, −20°C
  • 80% acetone
  • Lysis buffers (see recipe)
  • 400 pmol/µl CyDye DIGE fluors (see recipe)
  • 10 mM L‐lysine
  • IPG sample buffer (see recipe)
  • Ampholine sample buffer (see recipe)
  • Ice
  • 2 mM TCEP [tris(2‐carboxyethyl)phosphine; Thermo Scientific]
  • Centrifuge
  • 0.5‐ml microcentrifuge tubes
  • Vortex mixer
  • Low‐fluorescence glass gel plates
  • Gel fluorescence scanner (e.g., ProXPRESS, PE Biosystems or Typhoon 9000 series, GE Healthcare Life Sciences)
  • Image analysis software, such as:
    • ImageMaster2D, DeCyder (GE Healthcare)
    • Progenesis (Nonlinear Dynamics)
    • MELANIE (Geneva Bioinformatics)
    • AlphaMatch 2D (Alpha Innotech)
    • PDQuest (Bio‐Rad Laboratories)
    • Delta2D (Decodon)
    • Dymension (Syngene)
  • Additional reagents and equipment for 2‐D PAGE (unit )
NOTE: For comparison of different imaging software, see Kang et al. ( )

Support Protocol 1: Preparation of Protein for CY Labeling Reactions

  Materials
  • Cultured cells, tissue samples, whole organisms, and biofluids (specific examples are given in the text below)
  • Wash solution: 10 mM Tris·Cl, pH 8.0 ( ), and 5 mM magnesium acetate
  • Lysis buffer (see recipe; see Table 22.2.2)
  • Phenol saturated with TE (Sigma‐Aldrich)
  • TE saturated with phenol (see recipe)
  • Cell wash buffer: 75 mM phosphate‐buffered saline (PBS; )
  • 0.9% (w/v) saline
  • Centrifuge
  • Vortex mixer
  • Additional reagents and equipment for protein precipitation (see protocol 1Basic Protocol)
Table 2.2.2   MaterialsRecommended Lysis Buffers for 2‐D DIGE Sample Preparation a

Lysis buffer a Reagent Concentration
Standard b
CHAPS 4% (w/v)
Urea 8 M
Tris·Cl (pH 8.0‐9.0 for minimal dyes, pH 8.0 for saturation dyes) 10‐30 mM
Magnesium acetate 5 mM
Thiourea‐containing b
CHAPS 4% (w/v)
Urea 7 M
Thiourea 2 M
Tris·Cl (pH 8.0‐9.0 for minimal dyes, pH 8.0 for saturation dyes) 10‐30 mM
Magnesium acetate 5 mM
ASB14‐containing b
ASB14 c 2% (w/v)
Urea 7 M
Thiourea 2 M
Tris·Cl (pH 8.0‐9.0 for minimal dyes, pH 8.0 for saturation dyes) 10‐30 mM
Magnesium acetate 5 mM
SDS‐containing d
SDS 2% (w/v)
Tris·Cl (pH 8.0‐9.0 for minimal dyes, pH 8.0 for saturation dyes) 10‐30 mM
Magnesium acetate 5 mM

 aIt is advisable to consider adding protease inhibitors to the lysis buffer.
 bDo not heat solutions containing urea and thiourea in order to aid solubilization.
 cASB14 can be substituted with NP40 or SB3‐10, providing the final concentration of NP40 before labeling is <1%. Use 2% SB3‐10.
 dSDS‐containing solutions must be diluted to a final concentration of ≤0.2% before successful isoelectric focusing can take place.
NOTE: Typical cell lysing conditions involve resuspending a cell pellet in an appropriate lysis buffer and incubating for 30 to 60 min on ice. A typical cell washing procedure involves resuspending a cell pellet in wash solution at ≥2× the volume of the cell pellet, gently vortexing, and then re‐centrifuging.
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Figures

Videos

Literature Cited

Literature Cited
   Bruschi, M. 2009. New iodo‐acetamido cyanines for labeling cysteine thiol residues. A strategy for evaluating plasma proteins and their oxido‐redox status. Proteomics 9:460‐469.
   Chouchani, E.T. , Hurd, T.R. , Nadtochiy, S.M. , Brookes, P.S. , Fearnley, I.M. , Lilley, K.S. , Smith, R.A.J. , and Murphy, M.P. 2010. Identification of S‐nitrosated mitochondrial proteins by S‐nitrosothiol difference in gel electrophoresis (SNO‐DIGE): Implications for the regulation of mitochondrial function by reversible S‐nitrosation. Biochem. J. 430:49‐59.
   Chouchani, E.T. , James, A.M. , Fearnley, I.M. , Lilley, K.S. , and Murphy, M.P. 2011. Proteomic approaches to the characterization of protein thiol modification. Curr. Opin. Chem. Biol. 15:120‐128.
   Deery, M.J. , Maywood, E.S. , Chesham, J.E. , Slajdek, M. , Karp, N.A. , Green, E.W. , Charles, P.D. , Reddy, A.B. , Kyriacou, C.P. , Lilley, K.S. , and Hastings, M.J. 2009. Proteomic analysis reveals the role of synaptic vesicle cycling in sustaining the suprachiasmatic circadian clock. Curr. Biol. 19:2031‐2036.
   Kang, Y. , Techanukul, T. , Mantalaris, A. , and Nagy, J.M. 2009. Comparison of three commercially available DIGE analysis software packages: Minimal user intervention in gel‐based proteomics. J. Proteome Res. 8:1077‐1084.
   Karp, N.A. , McCormick, P.S. , Russell, M.R. , and Lilley, K.S. 2007. Experimental and statistical considerations to avoid false conclusions in proteomics studies using differential in‐gel electrophoresis. Mol. Cell. Proteomics 6:1354‐1364.
   Karp, N.A. , Feret, R. , Rubtsov, D.V. , and Lilley, K.S. 2008. Comparison of DIGE and post‐stained gel electrophoresis with both traditional and SameSpots analysis for quantitative proteomics. Proteomics 8:948‐960.
   Lilley, K.S. and Friedman, D.B. 2004. All about DIGE: Quantification technology for differential‐display 2D‐gel proteomics. Exp. Rev. Proteomics 1:401‐409.
   Lilley, K.S. , Razzaq, A. , and Dupree, P. 2002. Two‐dimensional gel electrophoresis: Recent advances in sample preparation, detection and quantitation. Curr. Opin. Chem. Biol. 6:46‐50.
   Minden, J.S. 2012. DIGE: Past and future. Methods Mol. Biol. 854:3‐8.
   Penno, M.A.S. , Klingler‐Hoffmann, M. , Brazzatti, J.A. , Boussioutas, A. , Putoczki, T. , Ernst, M. , and Hoffmann, P. 2012. 2D‐DIGE analysis of sera from transgenic mouse models reveals novel candidate protein biomarkers for human gastric cancer. J. Proteomics 77:40‐58.
   Santoni, V. , Kieffer, S. , Desclaux, D. , Masson, F. , and Rabilloud, T. 2000. Membrane proteomics: Use of additive main effects with multiplicative interaction model to classify plasma membrane proteins according to their solubility and electrophoretic properties. Electrophoresis 21:3329‐3344.
   Schmidt, H. , Gelhaus, C. , Latendorf, T. , Nebendahl, M. , Petersen, A. , Krause, S. , Leippe, M. , Becker, W.‐M. , and Janssen, O. 2009. 2‐D DIGE analysis of the proteome of extracts from peanut variants reveals striking differences in major allergen contents. Proteomics 9:3507‐3521.
   Shaw, J. , Rowlinson, R. , Nickson, J. , Stone, T. , Sweet, A. , Williams, K. , and Tonge, R. 2003. Evaluation of saturation labelling two‐dimensional difference gel electrophoresis fluorescent dyes. Proteomics 3:1181‐1195.
   Ünlü, M. , Morgan, M.E. , and Minden, J.S. 1997. Difference gel electrophoresis: A single gel method for detecting changes in protein extracts. Electrophoresis 18:2071‐2077.
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