Skip to main content
SpringerLink
Log in

Two-Dimensional Difference Gel Electrophoresis

  • Protocol
  • First Online:
Electrophoretic Separation of Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1855))

Abstract

Two-dimensional difference gel electrophoresis (2D DIGE) is a modified form of 2D electrophoresis (2D E) that allows one to compare two or three protein samples simultaneously on the same gel. The proteins in each sample are covalently tagged with different color fluorescent dyes that are designed to have no effect on the relative migration of proteins during electrophoresis. Proteins that are common to the samples appear as “spots” with a fixed ratio of fluorescent signals, whereas proteins that differ between the samples have different fluorescence ratios. With conventional imaging systems, DIGE is capable of reliably detecting as little as 0.2 fmol of protein, and protein differences down to ± 15%, over a ~10,000-fold protein concentration range. DIGE combined with digital image analysis therefore greatly improves the statistical assessment of proteome variation. Here we describe a protocol for conducting DIGE experiments, which takes 2–3 days to complete. We have further improved upon 2D DIGE by introducing in-gel equilibration to improve protein retention during transfer between the first and second dimensions of electrophoresis and by developing a fluorescent gel imaging system with a millionfold dynamic range.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 189.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. O’Farrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250:4007–4021

    PubMed  PubMed Central  Google Scholar 

  2. Scheele GA (1975) Two-dimensional gel analysis of soluble proteins. Characterization of guinea pig exocrine pancreatic proteins. J Biol Chem 250:5375–5385

    CAS  PubMed  Google Scholar 

  3. Klose J (1975) Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues. A novel approach to testing for induced point mutations in mammals. Humangenetik 26:231–243

    CAS  PubMed  Google Scholar 

  4. Gorg A, Postel W, Gunther S (1988) The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 9:531–546

    Article  CAS  Google Scholar 

  5. Hamdan M, Righetti PG (2005) Proteomics today: protein assessment and biomarkers using mass spectrometry, 2D electrophoresis, and microarray technology. John Wiley & Sons, Hoboken, N.J.

    Book  Google Scholar 

  6. Miller MJ, Olson AD, Thorgeirsson SS (1984) Computer analysis of two-dimensional gels: automatic matching. Electrophoresis 5:297–303

    Article  CAS  Google Scholar 

  7. Han CM, Park JH, Chang CS, Ryoo MC (2014) A spot-matching method using cumulative frequency matrix in 2D gel images. Biotechnol Biotechnol Equip 28:S37–S43

    Article  Google Scholar 

  8. Dowsey AW, Morris JS, Gutstein HB, Yang GZ (2010) Informatics and Statistics for Analyzing 2-D Gel Electrophoresis Images. Methods Mol Biol 604:239–255

    Article  CAS  Google Scholar 

  9. Ünlü M, Morgan ME, Minden JS (1997) Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 18:2071–2077

    Article  Google Scholar 

  10. Viswanathan S, Ünlü M, Minden JS (2006) Two-dimensional difference gel electrophoresis. Nat Protoc 1:1351–1358

    Article  CAS  Google Scholar 

  11. Lilley KS, Friedman DB (2004) All about DIGE: quantification technology for differential-display 2D-gel proteomics. Expert Rev Proteomics 1:401–409

    Article  CAS  Google Scholar 

  12. Gong L, Puri M, Unlu M, Young M, Robertson K, Viswanathan S, Krishnaswamy A, Dowd SR, Minden JS (2004) Drosophila ventral furrow morphogenesis: a proteomic analysis. Development 131:643–656

    Article  CAS  Google Scholar 

  13. Shaw MM, Riederer BM (2003) Sample preparation for two-dimensional gel electrophoresis. Proteomics 3:1408–1417

    Article  CAS  Google Scholar 

  14. Lilley K (2002) Protein profiling using two-dimensional difference gel electrophoresis (2-D DIGE). In: Coligan JE, Dunn BM, Speicher DW, Wingfield PT (eds) Current protocols in protein science. John Wiley & Sons, Inc., New York, pp 22.22.21–22.22.14

    Google Scholar 

  15. Marouga R, David S, Hawkins E (2005) The development of the DIGE system: 2D fluorescence difference gel analysis technology. Anal Bioanal Chem 382:669–678

    Article  CAS  Google Scholar 

  16. Wang H, Clouthier SG, Galchev V, Misek DE, Duffner U, Min CK, Zhao R, Tra J, Omenn GS, Ferrara JL, Hanash SM (2005) Intact-protein-based high-resolution three-dimensional quantitative analysis system for proteome profiling of biological fluids. Mol Cell Proteomics 4:618–625

    Article  CAS  Google Scholar 

  17. Okano T, Kondo T, Kakisaka T, Fujii K, Yamada M, Kato H, Nishimura T, Gemma A, Kudoh S, Hirohashi S (2006) Plasma proteomics of lung cancer by a linkage of multi-dimensional liquid chromatography and two-dimensional difference gel electrophoresis. Proteomics 6:3938–3948

    Article  CAS  Google Scholar 

  18. Diez R, Herbstreith M, Osorio C, Alzate O (2010) 2-D Fluorescence Difference Gel Electrophoresis (DIGE) in Neuroproteomics. In: Alzate O (ed) Neuroproteomics. CRC Press/Taylor & Francis, Boca Raton (FL)

    Google Scholar 

  19. Martyniuk CJ, Alvarez S, Denslow ND (2012) DIGE and iTRAQ as biomarker discovery tools in aquatic toxicology. Ecotoxicol Environ Saf 76:3–10

    Article  CAS  Google Scholar 

  20. Arentz G, Weiland F, Oehler MK, Hoffmann P (2015) State of the art of 2D DIGE. Proteomics Clin Appl 9:277–288

    Article  CAS  Google Scholar 

  21. Kondo T, Seike M, Mori Y, Fujii K, Yamada T, Hirohashi S (2003) Application of sensitive fluorescent dyes in linkage of laser microdissection and two-dimensional gel electrophoresis as a cancer proteomic study tool. Proteomics 3:1758–1766

    Article  CAS  Google Scholar 

  22. Sitek B, Potthoff S, Schulenborg T, Stegbauer J, Vinke T, Rump LC, Meyer HE, Vonend O, Stuhler K (2006) Novel approaches to analyse glomerular proteins from smallest scale murine and human samples using DIGE saturation labelling. Proteomics 6:4506–4513

    Article  Google Scholar 

  23. Zhou G, Li H, DeCamp D, Chen S, Shu H, Gong Y, Flaig M, Gillespie JW, Hu N, Taylor PR, Emmert-Buck MR, Liotta LA, Petricoin EF 3rd, Zhao Y (2002) 2D differential in-gel electrophoresis for the identification of esophageal scans cell cancer-specific protein markers. Mol Cell Proteomics 1:117–124

    Article  CAS  Google Scholar 

  24. Karp NA, Lilley KS (2005) Maximising sensitivity for detecting changes in protein expression: experimental design using minimal CyDyes. Proteomics 5:3105–3115

    Article  CAS  Google Scholar 

  25. Tonge R, Shaw J, Middleton B, Rowlinson R, Rayner S, Young J, Pognan F, Hawkins E, Currie I, Davison M (2001) Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology. Proteomics 1:377–396

    Article  CAS  Google Scholar 

  26. Yan JX, Devenish AT, Wait R, Stone T, Lewis S, Fowler S (2002) Fluorescence two-dimensional difference gel electrophoresis and mass spectrometry based proteomic analysis of Escherichia coli. Proteomics 2:1682–1698

    Article  CAS  Google Scholar 

  27. Van PT, Ganesan V, Bass V, Parthasarathy A, Schlesinger D, Minden JS (2014) In-gel equilibration for improved protein retention in 2DE-based proteomic workflows. Electrophoresis 35:3012–3017

    Article  CAS  Google Scholar 

  28. Sellers KF, Miecznikowski J, Viswanathan S, Minden JS, Eddy WF (2007) Lights, Camera, Action! systematic variation in 2-D difference gel electrophoresis images. Electrophoresis 28:3324–3332

    Article  CAS  Google Scholar 

  29. Van PT, Bass V, Shiwarski D, Lanni F, Minden J (2014) High dynamic range proteome imaging with the structured illumination gel imager. Electrophoresis 35:2642–2655

    Article  CAS  Google Scholar 

  30. Bertin E, Arnouts S (1996) SExtractor: software for source extraction. Astron Astrophys 117:393–404

    Google Scholar 

  31. Suehara Y, Kondo T, Fujii K, Hasegawa T, Kawai A, Seki K, Beppu Y, Nishimura T, Kurosawa H, Hirohashi S (2006) Proteomic signatures corresponding to histological classification and grading of soft-tissue sarcomas. Proteomics 6:4402–4409

    Article  CAS  Google Scholar 

  32. Shaw J, Rowlinson R, Nickson J, Stone T, Sweet A, Williams K, Tonge R (2003) Evaluation of saturation labelling two-dimensional difference gel electrophoresis fluorescent dyes. Proteomics 3:1181–1195

    Article  CAS  Google Scholar 

  33. Blundon MA, Schlesinger DR, Parthasarathy A, Smith SL, Kolev HM, Vinson DA, Kunttas-Tatli E, McCartney BM, Minden JS (2016) Proteomic analysis reveals APC-dependent post-translational modifications and identifies a novel regulator of beta-catenin. Development 143:2629–2640

    Article  CAS  Google Scholar 

  34. Millioni R, Miuzzo M, Sbrignadello S, Murphy E, Puricelli L, Tura A, Bertacco E, Rattazzi M, Iori E, Tessari P (2010) Delta2D and Proteomweaver: Performance evaluation of two different approaches for 2-DE analysis. Electrophoresis 31:1311–1317

    Article  CAS  Google Scholar 

  35. Goldfarb M (2007) Computer analysis of two-dimensional gels. J Biomol Tech 18:143–146

    PubMed  PubMed Central  Google Scholar 

  36. Zhou S, Bailey MJ, Dunn MJ, Preedy VR, Emery PW (2005) A quantitative investigation into the losses of proteins at different stages of a two-dimensional gel electrophoresis procedure. Proteomics 5:2739–2747

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The development and optimization of this DIGE protocol would not have been possible without the efforts and dedication of my students and staff: Mustafa Ünlü, Liz Morgan, Chris Lacenere, Surya Viswanathan, Lei Gong, Mamta Puri, Susan Dowd, and Amber Lucas, as well as current and past members of the undergraduate proteomics platoon.

Author information

Authors and Affiliations

  1. Department of Biological Science, Carnegie Mellon University, Pittsburgh, PA, USA

    Malachi Blundon, Vinitha Ganesan, Brendan Redler, Phu T. Van & Jonathan S. Minden

Authors
  1. Malachi Blundon
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Vinitha Ganesan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Brendan Redler
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Phu T. Van
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jonathan S. Minden
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jonathan S. Minden .

Editor information

Editors and Affiliations

  1. Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA

    Biji T. Kurien

  2. Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA

    R. Hal Scofield

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Blundon, M., Ganesan, V., Redler, B., Van, P.T., Minden, J.S. (2019). Two-Dimensional Difference Gel Electrophoresis. In: Kurien, B., Scofield, R. (eds) Electrophoretic Separation of Proteins. Methods in Molecular Biology, vol 1855. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8793-1_20

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-8793-1_20

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8792-4

  • Online ISBN: 978-1-4939-8793-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation