Advertisement

Two-Dimensional Electrophoresis: The State of the Art and Future Directions

  • Ben R. Herbert
  • Jean-Charles Sanchez
  • Luca Bini
Part of the Principles and Practice book series (PRINCIPLES)

Abstract

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is the only method currently available which is capable of simultaneously separating thousands of proteins. Thus 2-D PAGE is the heart of proteome technology. The first dimension of 2-D PAGE is isoelectric focusing (IEF), during which proteins are separated in a pH gradient until they reach a stationary position where their net charge is zero. The pH at which a protein has zero net charge is called its isoelectric point (pI). In the second dimension the proteins separated by IEF are separated orthogonally by electrophoresis in the presence of sodium dodecyl sulphate (SDSPAGE). The surfactant SDS binds to proteins, overriding their intrinsic charge, such that they all have the same charge density and free solution electrophoretic mobility. When the SDS-coated proteins migrate in a sieving polyacrylamide gel they separate based on their molecular mass. The high resolution of 2-D PAGE results from the first and second dimension separations being based on independent protein parameters.

Keywords

Carrier Ampholyte Increase Protein Solubility High Protein Load Page Separation Protein Prefractionation 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Anderson L, Seilhamer J (1997) A comparison of selected mRNA and protein abundances in human liver. Electrophoresis 18: 533–537PubMedCrossRefGoogle Scholar
  2. Anderson NL, Nance SL, Tollaksen SL, Giere FA, Anderson NG (1985) Quantitative reproducibility of measurements from Coomassie Blue-stained two-dimensional gels: analysis of mouse liver protein patterns and a comparison of BALB/c and C57 strains. Electrophoresis 6: 592–599CrossRefGoogle Scholar
  3. Anderson NL, Giere FA, Nance SL, Gemmell MA, Tollaksen SL, Anderson NG (1987) Effects of toxic agents at the protein level: quantitative measurement of 213 mouse liver proteins following xenobiotic treatment. Fundam Appl Toxicol 8: 39–50PubMedCrossRefGoogle Scholar
  4. Bier M, Egen NB, Allgyer TT, Twitty GE, Mosher RA (1979) In: Gross E, Meienhofer J (eds) Peptides: structure and biological functions. Pierce Chemical Co Rockford, Illinois, pp 79–89Google Scholar
  5. Bini L, Magi B, Marzocchi B, Cellesi C, Berti B, Raggiaschi R, Rossolini A, Pallini V (1996) Two-dimensional electrophoretic patterns of acute-phase human serum proteins in the course of bacterial and viral diseases. Electrophoresis 17: 612–616PubMedCrossRefGoogle Scholar
  6. Bjellqvist B, Ek K, Righetti PG, Gianazza E, Görg A, Westermeier R, Postel W (1982) Isoelectric focusing in immobilised pH gradients: principle, methodology and some applications. J Biochem Biophys Meth 6: 317–339PubMedCrossRefGoogle Scholar
  7. Bjellqvist B, Pasquali C, Ravier F, Sanchez JC, Hochstrasser D (1993a) A nonlinear wide-range immobilised pH gradient for two-dimensional electrophoresis and its definition in a relevant pH scale. Electrophoresis 14: 1357–1365PubMedCrossRefGoogle Scholar
  8. Bjellqvist B, Sanchez JC, Pasquali C, Ravier F, Paquet N, Frutiger S, Hughes GJ, Hochstrasser D (1993b) Micropreparative 2-D electrophoresis allowing the separation of milligram amounts of proteins. Electrophoresis 14: 1375–1378PubMedCrossRefGoogle Scholar
  9. Blomberg A, Blomberg L, Norbeck J, Fey SJ, Larsen PM, Larsen M, Roepstorff P, Degand H, Boutry M, Posch A, Görg A (1995) Interlaboratory reproducibility of yeast protein patterns analysed by immobilised pH gradient two-dimensional gel electrophoresis. Electrophoresis 16: 1935–1945PubMedCrossRefGoogle Scholar
  10. Bossi A, Gelfi C, Orsi A, Righetti PG (1994a) Isoelectric focusing of histones in extremely alkaline immobilised pH gradients: comparison with capillary electrophoresis. J Chromatogr A 686: 121–128CrossRefGoogle Scholar
  11. Bossi A, Righetti PG, Vecchio G, Severinsen S (1994b) Focusing of alkaline proteases (sub-tilisins) in pH 10–12 immobilised gradients. Electrophoresis 15: 1535–1540PubMedCrossRefGoogle Scholar
  12. Bossinger J, Miller MJ, Vo KP, Geiduschek EP, Xuong NH (1979) Quantitative analysis oftwo-dimensional electrophoretograms. J Biol Chem 254: 7986–7998PubMedGoogle Scholar
  13. Celis JE, Rasmussen HH, Madsen P, Leffers H, Honoré B, Dejgaard K, Gesser B, Olsen E, Gromov P, Hoffmann HJ, Nielsen M, Celis A, Basse B, Lauridsen JB, Ratz GP, Nielsen H, Andersen AH, Walbrum E, Kjaergaard I, Puype M, Van Damme J, Vandekerckhove J (1992) The human keratinocyte two-dimensional gel protein database (update 1992): towards an integrated approach to the study of cell proliferation, differentiation and skin diseases. Electrophoresis 13: 893–959PubMedCrossRefGoogle Scholar
  14. Celis, JE, Rasmussen HH, Gromov P, Olsen E, Madsen P, Leffers H, Honoré B, Dejgaard K, Vorum H, Kristensen DB, Ostergaard M, Haunso A, Jensen NA, Celis A, Basse B, Lauridsen J, Ratz GP, Andersen AH, Walbum E, Kjaergaard I, Andersen I, Puype M, VanGoogle Scholar
  15. Damme J, Vandekerckhove J (1995) The human keratinocyte two-dimensional gel protein database (update 1995): mapping components of signal transduction pathways. Electrophoresis 14: 1091–1098Google Scholar
  16. Corbett JM, Dunn MJ, Posch A, Görg A (1994) Positional reproducibility of protein spots in two-dimensional polyacrylamide electrophoresis using immobilised pH gradient isoelectric focusing in the first dimension — an interlaboratory comparison. Electrophoresis 15: 1205–1211PubMedCrossRefGoogle Scholar
  17. Corthals GL, Molloy MP, Herbert BR, Williams KL, Gooley AA (1997) Prefractionation of protein samples prior to two-dimensional electrophoresis. Electrophoresis 18: 317–323PubMedCrossRefGoogle Scholar
  18. Dunn MJ (1987) Two-dimensional polyacrylamide gel electrophoresis. In: Chrambach A, Dunn MJ, Radola BJ (eds) Advances in electrophoresis. VCH, Weinheim, Germany, pp 1–109Google Scholar
  19. Dunn MJ (1993) Gel electrophoresis: proteins. Bios Scientific Publishers, Oxford, UK Eckerskorn C, Strupat K, Schleuder D, Hochstrasser DF, Sanchez JC, Lottspeich FGoogle Scholar
  20. Hillenkamp F (1997) Analysis of proteins by direct scanning-infrared-MALDI mass spec-Google Scholar
  21. trometry after 2-D PAGE separation and electroblotting. Anal Chem in press Esteve-Romero J, Simo-Alfonso E, Bossi A, Bresciani F, Righetti PG (1996) Sample streaksGoogle Scholar
  22. and smears in immobilised pH gradient gels. Electrophoresis 17:704–708Google Scholar
  23. Garrels JI (1983) Quantitative two-dimensional gel electrophoresis of proteins. Methods Enzymol 100: 411–423PubMedCrossRefGoogle Scholar
  24. Giometti CS, Gemmell MA, Nance SL, Tollaksen SL, Taylor J (1987) Detection of heritable mutations as quantitative changes in protein expression. J Biol Chem 262: 12764–12767PubMedGoogle Scholar
  25. Giometti CS, Tollaksen SL, Gemmell MA, Taylor J, Hawes J, Roderick T (1990) The analysis of recessive lethal mutations in mice by using two-dimensional gel electrophoresis of liver proteins. Mut Res 242: 47–55CrossRefGoogle Scholar
  26. Giometti CS, Gemmell MA, Tollaksen SL, Taylor J (1991) Quantitation of human leukocyte proteins after silver staining: a study with two-dimensional electrophoresis. Electrophoresis 12: 536–543PubMedCrossRefGoogle Scholar
  27. Görg A, Postel W, Weser J, Gunther S, Strahler JR, Hanash SM, Sommerlot L (1987) Elimination of point streaking on silver stained two-dimensional gels by addition of iodoacetamide to the equilibration buffer. Electrophoresis 8: 122–124CrossRefGoogle Scholar
  28. Görg A, Postel W, Gunther S (1988) The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 9: 531–546PubMedCrossRefGoogle Scholar
  29. Görg A, Obermaier C, Boguth G, Csordas A, Diaz JJ, Madjar JJ (1997) Very alkaline immobilised pH gradients for two-dimensional electrophoresis of ribosomal and nuclear proteins. Electrophoresis 18: 328–337PubMedCrossRefGoogle Scholar
  30. Hanash SM, Strahler JR, Neel JV, Hailat N, Melhem R, Keim D, Zhu XX, Wagner D, Gage DA, Watson JT (1991) Highly resolving two-dimensional gels for protein sequencing. Proc Natl Acad Sci USA 88: 5709–5713PubMedCrossRefGoogle Scholar
  31. Herbert BR, Molloy MP, Gooley AA, Walsh BJ, Bryson WG, Williams KL (1997) Improvedprotein solubility in 2-D electrophoresis using tributyl phosphine. Submitted Hochstrasser DF, Merril CR (1988) “Catalysts” for polyacrylamide gel polymerization anddetection of proteins by silver staining. Appl Theor Electrophor 1: 35–40Google Scholar
  32. Huber LA, Pasquali C, Gagescu R, Zuk A, Gruenberg J, Matlin KS (1996) Endosomal fractions from viral K-ras-transformed MDCK cells reveal transformation specific changes on two-dimensional gel maps. Electrophoresis 17: 1734–1740PubMedCrossRefGoogle Scholar
  33. Jackson P, Urwin VE, Mackay CD (1988) Rapid imaging, using a cooled charged-coupleddevice, of fluorescent two-dimensional polyacrylamide gels produced by labelling proteins in the first dimensional isoelectric focusing gel with the fluorophore 2-methoxy-2,4diphenyl-3(2H)furanone. Electrophoresis 9: 330–339PubMedCrossRefGoogle Scholar
  34. Kenrick KG, Margolis J (1970) Isoelectric focusing and gradient gel electrophoresis: a two-dimensional technique. Anal Biochem 33: 204–207PubMedCrossRefGoogle Scholar
  35. Klose J (1975) Protein mapping by combined isoelectric focusing and electrophoresis in mouse tissues. A novel approach to testing for induced point mutations in mammals. Humangenetik 26: 231–243PubMedGoogle Scholar
  36. Liberatori S, Bini L, De Felice C, Magi B, Marzocchi B, Raggiaschi R, Pallini V, Bracci RGoogle Scholar
  37. (1997).
    Acute-phase proteins in perinatal human plasma. Electrophoresis 18:520–526 Matsudaira P (1987) Sequence from picomole quantities of proteins electroblotted ontoGoogle Scholar
  38. poly-vinylidene difluoride membranes. J Biol Chem 262:10035–10038Google Scholar
  39. Merril CR, Goldman D, Van Keuren ML (1984) Gel protein stains: silver stain. Methods Enzymol 104: 441–447PubMedCrossRefGoogle Scholar
  40. Merril CR, Creed GJ, Joy J, Olson AD (1993) Identification and use of constitutive proteins for the normalization of high resolution electrophoretograms. Appl Theor Electrophor 3: 329–333PubMedGoogle Scholar
  41. O’Farrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250: 4007–4021PubMedGoogle Scholar
  42. Rabilloud T (1994) Two-dimensional electrophoresis of basic proteins with equilibrium iso-electric focusing in carrier ampholyte pH gradients. Electrophoresis 15: 278–282PubMedCrossRefGoogle Scholar
  43. Rabilloud T (1996) Solubilisation of proteins for electrophoretic analyses. Electrophoresis17: 813–829Google Scholar
  44. Rabilloud T, Hubert M, Tarroux P (1986) Procedures for two-dimensional electrophoretic analysis of nuclear proteins. J Chromatogr 351: 77–89CrossRefGoogle Scholar
  45. Rabilloud T, Valette C, Lawrence JJ (1994) Sample application by in-gel rehydration improves the resolution of two-dimensional electrophoresis with immobilised pH gradients in the first dimension. Electrophoresis 15: 1552–1558PubMedCrossRefGoogle Scholar
  46. Rabilloud T, Adessi C, Giraudel A, Lunardi J (1997) Improvement of the solubilisation of proteins in two-dimensional electrophoresis with immobilised pH gradients. Electrophoresis 18: 307–316PubMedCrossRefGoogle Scholar
  47. Righetti PG (1990) Immobilised pH gradients: theory and methodology. In: Burdon RH, van Knippenberg PH (eds) Laboratory techniques in biochemistry and molecular biology. Elsevier, AmsterdamGoogle Scholar
  48. Righetti PG, Wenisch E, Faupel M (1989) Preparative protein purification in a multi-compartment electrolyser with Immobiline membranes. J Chromatogr 475: 293–309CrossRefGoogle Scholar
  49. Sanchez JC, Paquet N, Hughes GJ, Hochstrasser DF (1992) Preparative 2-D purifies proteins for sequencing or antibody production. US/EG Bio-Rad Bulletin 1744Google Scholar
  50. Sanchez JC, Rouge V, Pisteur M, Ravier F, Tonella L, Moosmayer M, Wilkins MR, Hochstrasser DF (1997a) Improved and simplified in-gel sample application using reswelling of dry immobilised pH gradients. Electrophoresis 18: 324–327PubMedCrossRefGoogle Scholar
  51. Sanchez JC, Wirth P, Jaccoud S, Appel RD, Sarto C, Wilkins MR, Hochstrasser DF (1997b) Simultaneous analysis of cyclin and oncogene expression using multiple monoclonal antibody immunoblots. Electrophoresis 18: 638–641PubMedCrossRefGoogle Scholar
  52. Scheele GA (1975) Two-dimensional gel analysis of soluble proteins. Characterisation of guinea pig exocrine pancreatic proteins. J Biol Chem 250: 5375–5385PubMedGoogle Scholar
  53. Steinberg TH, Haugland RP, Singer VL (1996) Applications of SYPRO Orange and SYPRO Red protein gel stains. Anal Biochem 239: 238–245PubMedCrossRefGoogle Scholar
  54. Tal M, Silberstein A, Nusser E (1985), Why does Coomassie Brilliant Blue R interact differently with different proteins? J Biol Chem 260: 9976–9980PubMedGoogle Scholar
  55. Wilkins MR, Sanchez JC, Williams KL, Hochstrasser DF (1996) Current challenges and future applications for protein maps and post-translational vector maps in proteome projects. Electrophoresis 17: 830–838PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 1997

Authors and Affiliations

  • Ben R. Herbert
  • Jean-Charles Sanchez
  • Luca Bini

There are no affiliations available

Personalised recommendations