Two-Dimensional Difference Gel Electrophoresis: A Gel-Based Proteomic Approach for Protein Analysis

  • Weimin GaoEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2102)


Two-dimensional difference gel electrophoresis (2D-DIGE) remains to be one of the most popular and versatile methods of protein separation among many proteomics technologies. Similar to traditional two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), the proteins are separated based on their charges and molecular weight by 2D-DIGE. Different from 2D-PAGE, proteins are pre-labeled with different fluorescent dyes, and different protein samples are run in one gel by this method. Therefore, 2D-DIGE not only carries the advantages of 2D-PAGE but also eliminates gel-to-gel variation and achieves high resolution, sensitivity, and reproducibility.

Key words

Two-dimensional difference gel electrophoresis Two-dimensional polyacrylamide gel electrophoresis Protein separation Proteomics 


  1. 1.
    Kenyon GL, DeMarini DM, Fuchs E, Galas DJ, Kirsch JF, Leyh TS, Moos WH, Petsko GA, Ringe D, Rubin GM, Sheahan LC (2002) Defining the mandate of proteomics in the post-genomics era: workshop report. Mol Cell Proteomics 1(10):763–780PubMedGoogle Scholar
  2. 2.
    Hochstrasser DF, Sanchez JC, Appel RD (2002) Proteomics and its trends facing nature’s complexity. Proteomics 2(7):807–812CrossRefGoogle Scholar
  3. 3.
    Gygi SP, Corthals GL, Zhang Y, Rochon Y, Aebersold R (2000) Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology. Proc Natl Acad Sci U S A 97(17):9390–9395CrossRefGoogle Scholar
  4. 4.
    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(3):231–243PubMedGoogle Scholar
  5. 5.
    O’Farrell PZ, Goodman HM, O’Farrell PH (1977) High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 12(4):1133–1141CrossRefGoogle Scholar
  6. 6.
    Espina V, Mehta AI, Winters ME, Calvert V, Wulfkuhle J, Petricoin EF 3rd, Liotta LA (2003) Protein microarrays: molecular profiling technologies for clinical specimens. Proteomics 3(11):2091–2100CrossRefGoogle Scholar
  7. 7.
    Grubb RL, Calvert VS, Wulkuhle JD, Paweletz CP, Linehan WM, Phillips JL, Chuaqui R, Valasco A, Gillespie J, Emmert-Buck M, Liotta LA, Petricoin EF (2003) Signal pathway profiling of prostate cancer using reverse phase protein arrays. Proteomics 3(11):2142–2146CrossRefGoogle Scholar
  8. 8.
    Tang N, Tornatore P, Weinberger SR (2004) Current developments in SELDI affinity technology. Mass Spectrom Rev 23(1):34–44CrossRefGoogle Scholar
  9. 9.
    Templin MF, Stoll D, Schwenk JM, Potz O, Kramer S, Joos TO (2003) Protein microarrays: promising tools for proteomic research. Proteomics 3(11):2155–2166CrossRefGoogle Scholar
  10. 10.
    Haab BB (2003) Methods and applications of antibody microarrays in cancer research. Proteomics 3(11):2116–2122CrossRefGoogle Scholar
  11. 11.
    Hanash S (2003) The emerging field of protein microarrays. Proteomics 3(11):2075CrossRefGoogle Scholar
  12. 12.
    Graslund S, Falk R, Brundell E, Hoog C, Stahl S (2002) A high-stringency proteomics concept aimed for generation of antibodies specific for cDNA-encoded proteins. Biotechnol Appl Biochem 35(Pt 2):75–82CrossRefGoogle Scholar
  13. 13.
    James P (2002) Chips for proteomics: a new tool or just hype? BioTechniques 4-10:12–13Google Scholar
  14. 14.
    Kusnezow W, Hoheisel JD (2002) Antibody microarrays: promises and problems. BioTechniques Suppl:14–23CrossRefGoogle Scholar
  15. 15.
    Moody MD, Van Arsdell SW, Murphy KP, Orencole SF, Burns C (2001) Array-based ELISAs for high-throughput analysis of human cytokines. BioTechniques 31(1):186–190, 192-194CrossRefGoogle Scholar
  16. 16.
    O'Farrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250(10):4007–4021PubMedPubMedCentralGoogle Scholar
  17. 17.
    Garcia A (2007) Two-dimensional gel electrophoresis in platelet proteomics research. Methods Mol Med 139:339–353CrossRefGoogle Scholar
  18. 18.
    Carrette O, Burkhard PR, Sanchez JC, Hochstrasser DF (2006) State-of-the-art two-dimensional gel electrophoresis: a key tool of proteomics research. Nat Protoc 1(2):812–823CrossRefGoogle Scholar
  19. 19.
    Gorg A, Weiss W, Dunn MJ (2004) Current two-dimensional electrophoresis technology for proteomics. Proteomics 4(12):3665–3685. Scholar
  20. 20.
    Unlu M, Morgan ME, Minden JS (1997) Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 18(11):2071–2077. Scholar
  21. 21.
    Lathrop JT, Anderson NL, Anderson NG, Hammond DJ (2003) Therapeutic potential of the plasma proteome. Curr Opin Mol Ther 5(3):250–257PubMedGoogle Scholar
  22. 22.
    Pieper R, Gatlin CL, Makusky AJ, Russo PS, Schatz CR, Miller SS, Su Q, McGrath AM, Estock MA, Parmar PP, Zhao M, Huang ST, Zhou J, Wang F, Esquer-Blasco R, Anderson NL, Taylor J, Steiner S (2003) The human serum proteome: display of nearly 3700 chromatographically separated protein spots on two-dimensional electrophoresis gels and identification of 325 distinct proteins. Proteomics 3(7):1345–1364CrossRefGoogle Scholar
  23. 23.
    Gao W, Lu C, Kochanek PM, Berger RP (2014) Serum amyloid A is increased in children with abusive head trauma: a gel-based proteomic analysis. Pediatr Res 76(3):280–286. Scholar
  24. 24.
    Liu Z, Feng D, Gu D, Zheng R, Esperat C, Gao W (2017) Differentially expressed haptoglobin as a potential biomarker for type 2 diabetic mellitus in Hispanic population. Biofactors 43(3):424–433. Scholar
  25. 25.
    VanBogelen RA (1999) Generating a bacterial genome inventory. Identifying 2-D spots by comigrating products of the genome on 2-D gels. Methods Mol Biol 112:423–429PubMedGoogle Scholar
  26. 26.
    Karuppusamy S, Mutharia L, Kelton D, Karrow N, Kirby G (2018) Identification of antigenic proteins from Mycobacterium avium subspecies paratuberculosis cell envelope by comparative proteomic analysis. Microbiology 164(3):322–337. Scholar
  27. 27.
    Jang JH, Hanash S (2003) Profiling of the cell surface proteome. Proteomics 3(10):1947–1954CrossRefGoogle Scholar
  28. 28.
    Li KW, Hornshaw MP, Van Der Schors RC, Watson R, Tate S, Casetta B, Jimenez CR, Gouwenberg Y, Gundelfinger ED, Smalla KH, Smit AB (2004) Proteomics analysis of rat brain postsynaptic density. Implications of the diverse protein functional groups for the integration of synaptic physiology. J Biol Chem 279(2):987–1002CrossRefGoogle Scholar
  29. 29.
    Garrels JI (1979) Two dimensional gel electrophoresis and computer analysis of proteins synthesized by clonal cell lines. J Biol Chem 254(16):7961–7977PubMedGoogle Scholar
  30. 30.
    Anderson NG, Anderson NL (1996) Twenty years of two-dimensional electrophoresis: past, present and future. Electrophoresis 17(3):443–453. Scholar
  31. 31.
    Minden JS, Dowd SR, Meyer HE, Stuhler K (2009) Difference gel electrophoresis. Electrophoresis 30(Suppl 1):S156–S161. Scholar
  32. 32.
    Van den Bergh G, Arckens L (2004) Fluorescent two-dimensional difference gel electrophoresis unveils the potential of gel-based proteomics. Curr Opin Biotechnol 15(1):38–43. Scholar
  33. 33.
    Lilley KS, Friedman DB (2006) Difference gel electrophoresis DIGE. Drug Discov Today Technol 3(3):347–353CrossRefGoogle Scholar
  34. 34.
    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(7):1181–1195. Scholar
  35. 35.
    Viswanathan S, Unlu M, Minden JS (2006) Two-dimensional difference gel electrophoresis. Nat Protoc 1(3):1351–1358CrossRefGoogle Scholar
  36. 36.
    Tannu NS, Hemby SE (2006) Two-dimensional fluorescence difference gel electrophoresis for comparative proteomics profiling. Nat Protoc 1(4):1732–1742CrossRefGoogle Scholar
  37. 37.
    Shao C, Chen L, Lu C, Shen CL, Gao W (2011) A gel-based proteomic analysis of the effects of green tea polyphenols on ovariectomized rats. Nutrition 27(6):681–686. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of Occupational and Environmental Health Sciences, School of Public HealthWest Virginia UniversityMorgantownUSA

Personalised recommendations