Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Classic Protocol
  • Published:

Two-dimensional gel electrophoresis of proteins

Two-dimensional polyacrylamide gel electrophoresis (PAGE) is used for separation of complex protein mixtures by the independent parameters of isoelectric point and molecular weight. Isoelectric focusing (IEF) separates proteins in a pH gradient. Each protein is 'focused' because it moves under the influence of the electric field until it reaches its isoelectric point, the pH at which it has no net charge. After IEF in the presence of urea and a nonionic detergent, the IEF gel is equilibrated in sodium dodecyl sulfate (SDS) to prepare the proteins for SDS-PAGE. The method described here1 uses carrier ampholytes to form a pH gradient in a long, thin (1.2-mm) focusing gel composed of a low percentage (2.7%) of acrylamide and containing 9.5 M urea and 2% Nonidet P-40 to maintain protein solubility. After IEF, the gel is briefly equilibrated in SDS and placed directly onto the top edge of a second-dimension slab gel. Because the time between the end of IEF and the start of SDS-PAGE is only a few minutes (thereby minimizing diffusion), the spots are highly resolved and nearly round in shape.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

References

  1. Garrels, J.I. Two-dimensional gel electrophoresis and computer analysis of proteins synthesized by clonal cell lines. J. Biol. Chem. 254, 7961–7977 (1979).

    CAS  PubMed  Google Scholar 

  2. Garrels, J.I. Quantitative two-dimensional gel electrophoresis of proteins. Methods Enzymol. 100, 411–423 (1983).

    Article  CAS  Google Scholar 

Download references

Rights and permissions

Reprints and permissions

About this article

Cite this article

Two-dimensional gel electrophoresis of proteins. Nat Methods 2, 83–84 (2005). https://doi.org/10.1038/nmeth0105-83

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth0105-83

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing