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Two-Dimensional Gel Electrophoresis: Vertical Isoelectric Focusing

  • Yaser Dorri
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1855)

Abstract

Two-dimensional gel electrophoresis is one of the most powerful tools for separating proteins based on their size and charge. Two-dimensional gel electrophoresis (2-DE) is very useful to separate two proteins with identical molecular weights but different charges, which cannot be achieved with just sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Here, a simpler and easier version of 2-DE is presented which is also faster than all the currently available techniques. In this modified version of 2-DE, isoelectric focusing is carried out in the first dimension using a vertical sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) apparatus. Following the first-dimensional IEF, each individual lane is excised from the IEF gel and after a 90° rotation, is inserted into a second-dimensional SDS-PAGE, which can be stained with Coomassie Brilliant Blue for protein analysis or immunoblotted for further analysis. This version of IEF can be run in less than 2 h compared to the overnight run required by O’Farrell’s method. Difficult tube gel casting and gel extrusion as well as tube gel distortion are eliminated in our method. This method is simpler, faster, and inexpensive. Both dimensions can be done on the same SDS-PAGE apparatus, and up to ten samples can be run simultaneously using one gel.

Key words

2D gel electrophoresis Isoelectric focusing Sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS-PAGE 2-DE IEF 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Yaser Dorri
    • 1
    • 2
    • 3
  1. 1.Arthritis and Clinical ImmunologyOklahoma Medical Research FoundationOklahoma CityUSA
  2. 2.Department of Medicine EndocrinologyUniversity of Oklahoma Health Sciences CenterOklahoma CityUSA
  3. 3.University of OklahomaNormanUSA

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