Large-Format 2-D Polyacrylamide Gel Electrophoresis

  • Henry Brzeski
  • Stephen Russell
  • Anthony G. Sullivan
  • Richard I. Somiari
  • Craig D. Shriver
Part of the Springer Protocols Handbooks book series (SPH)


Proteins are composed of different numbers of neutral positively and negatively charged amino acids. Therefore, proteins vary widely in size and have either positive, negative, or zero net charge, depending on the pH of their surroundings. The original two-dimensional gel electrophoresis format was developed almost 30 years ago (1) to exploit this variation in protein charge and size for separation purposes. The isoelectric point of a protein (pI) is the pH at which it has a net zero charge, which, for the majority of proteins, lies between pH 4.0 and 8.0. The sizes of proteins vary widely (10–500 kDa), with an average molecular weight of approx 50 kDa. These two mutually independent properties are exploited by firstly denaturing proteins in urea and then subjecting them to an electric field in a pH gradient established in a low-concentration polyacrylamide gel (originally this pH gradient was formed, using ampholytes, in situ, but now precast immobilized pH gradient [IPG] strips [2, 3, 4] are found to be more reliable). In this case, all but the very largest proteins can migrate freely until they reach a pH at which they have no net charge (isoelectric focusing [IEF]). After completion of the focusing, the proteins are denatured in situ, their native charge is saturated with the anionic detergent sodium dodecyl sulphate (SDS), and then the gel is layered, perpendicular to the direction of focusing, on a higher-concentration polyacrylamide gel, and the focused proteins are separated on the basis of size. This gives rise to discrete spots representing one (or perhaps a very small number) of different proteins.


Sodium Dodecyl Sulphate Anionic Detergent Sodium Dodecyl Sulphate Acrylamide Solution Strip Holder Acrylamide Polymerization 
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Copyright information

© Humana Press Inc., Totowa, NJ 2005

Authors and Affiliations

  • Henry Brzeski
    • 3
  • Stephen Russell
    • 3
  • Anthony G. Sullivan
    • 2
    • 1
  • Richard I. Somiari
    • 4
  • Craig D. Shriver
    • 5
  1. 1.Functional Genomics and Proteomics Unit, Windber Research InstituteWindber
  2. 2.Functional Genomics and Proteomics Unit, Thermoelectron Training InstituteWest Palm Beach
  3. 3.Functional Genomics and Proteomics Unit, Windber ResearchInstituteWindber
  4. 4.Functional Genomics and Protemics Unit, ITSI-BiosciencesJohnstown
  5. 5.Clinical Breast Care Project, Walter Reed Army Medical CenterWashington,DC

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