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State-of-the-art two-dimensional gel electrophoresis: a key tool of proteomics research

Nature Protocols volume 1, pages 812–823 (2006)Cite this article

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Abstract

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is the most popular and versatile method of protein separation among a rapidly growing array of proteomics technologies. Based on two distinct procedures, it combines isoelectric focusing (IEF), which separates proteins according to their isoelectric point (pI), and SDS-PAGE, which separates them further according to their molecular mass. At present, 2D-PAGE is capable of simultaneously detecting and quantifying up to several thousand protein spots in the same gel image. Here we provide comprehensive step-by-step instructions for the application of a standardized 2D-PAGE protocol to a sample of human plasma or cerebrospinal fluid (CSF). The method can be easily adapted to any type of sample. This four-day protocol provides detailed information on how to apply complex biological fluids to an immobilized dry strip gel, cast home-made gradient acrylamide gels, run the gels, and perform standard staining methods. A troubleshooting guide is also included.

NOTE:The version of this article initially published online contained the following errors in the REAGENT SETUP section on p. 814: Under “Strip reswelling buffer”, “0.0375 g of DTE” should be “0.0385 g of DTE”. Under “Iso Buffer”, “5.4 g of urea” should be “4.8 g of urea”. Under “Electrophoresis buffer, upper tank”, “18 g of Tris” should be “9.09 g of Tris” and “86.4 g of glycine” should be “44.6 g of glycine”. Under “Electrophoresis buffer, lower tank”, “90 g of Tris” should be “45.45 g of Tris” and “432 g of glycine” should be “222.9 g of glycine”. Under “Agarose sealing solution”, “5%” should be “0.5%”. Under “Sensitizer solution”, “68.4 g of sodium acetate” should be “68.04 g of sodium acetate trihydrate”. Under “Ammoniacal silver nitrate solution”, “Silver nitrate” should be “47 mM silver nitrate”; “10 N sodium hydroxide” should be “0.02 N sodium hydroxide”; and “25% ammonium hydroxide (27% pure, Merck)” should be 0.33% ammonium hydroxide (25% pure, Merck)”. The sentence “Add 6 g of silver nitrate dissolved in dH2O to 30ml slowly into a solution containing 160 ml of dH2O, 10 ml of concentrated ammonia (25%) and 1.5 ml of 10 N sodium hydroxide” should read “Dissolve 6 g of silver nitrate in dH2O to 30 ml, then add slowly into a solution containing 160 ml of dH2O, 10 ml of 25% ammonium hydroxide and 1.5 ml of 10 N sodium hydroxide.” In Table 1 (p. 816), “Up to 100,000 kVh” should be “100kVh”. These errors have been corrected in all versions of the article.

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Figure 1
Figure 2: 2D-PAGE gel of human plasma stained with sensitive silver.
Figure 3: 2D-PAGE gel of human CSF.
Figure 4: Sample loading troubleshooting.
Figure 5: Horizontal smearing of proteins.
Figure 6: Distortion along the second dimension.
Figure 7: Gel preparation troubleshooting.
Figure 8: 2D-PAGE gel of mouse brain sample showing disparities in silver staining.

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  • 22 March 2007

    see erratum

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Acknowledgements

Many colleagues have contributed to the development of this protocol including B. Bjellqvist, V. Converset, M. Côte, O. Golaz, C. Pasquali, F. Ravier, N. Rodrigo, L. Tonella and C. Zimmermann.

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Authors and Affiliations

  1. Department of Structural Biology and Bioinformatics, Biomedical Proteomics Research Group, Faculty of Medicine, Geneva University, 1 rue Michel Servet, Geneva 4, CH-1211, Switzerland

    Odile Carrette, Pierre R Burkhard, Jean-Charles Sanchez & Denis F Hochstrasser

  2. Neurology Department, Geneva University Hospital, 24 rue Micheli-du-Crest, Geneva 14, CH-1211, Switzerland

    Pierre R Burkhard

  3. Central Clinical Chemistry Laboratory, Geneva University Hospital, 24 rue Micheli-du-Crest, Geneva 14, CH-1211, Switzerland

    Denis F Hochstrasser

  4. Pharmacy Section, Faculty of Sciences, Geneva University, 30 quai Ernest-Ansermet, Geneva 4, CH-1211, Switzerland

    Denis F Hochstrasser

Authors
  1. Odile Carrette
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  2. Pierre R Burkhard
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  4. Denis F Hochstrasser
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Contributions

O.C. prepared the manuscript and optimized SyproRuby fluorescent staining; P.R.B. optimized 2D-PAGE of CSF samples; J.-C.S. and D.F.H. developed the 2D-PAGE protocol.

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Correspondence to Denis F Hochstrasser.

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Carrette, O., Burkhard, P., Sanchez, JC. et al. State-of-the-art two-dimensional gel electrophoresis: a key tool of proteomics research. Nat Protoc 1, 812–823 (2006). https://doi.org/10.1038/nprot.2006.104

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