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Keywords:

  • Gel electrophoresis;
  • serum proteomics;
  • sample preparation;
  • protein fractionation;
  • TCA precipitation

Abstract

Two-dimensional gel electrophoresis (2DGE) remains an important tool in the study of biological systems by proteomics. While the use of 2DGE is commonplace in research publications, there are few instructional laboratories that address the use of 2DGE for analyzing complex protein samples. One reason for this lack is the fact that the preparation of samples for 2DGE is a complex and difficult process that can commonly yield gels of poor quality and resolution. In this experiment, we use a serum-based sample to mitigate many of the sample preparation issues that occur in cell-based sample preparations and incorporate a protein precipitation method that was developed to address the problem of high-abundance proteins and dynamic range in serum proteomics research. By focusing on 2DGE apart from many other facets of proteomic experimental design, students have the opportunity to gain fruitful experience in the use of this workhorse proteomics technique. This simplified focus also makes this exercise accessible to biochemistry instructors who are not active in proteomics; the requisite techniques may require some new equipment (i.e. an isoelectric focusing apparatus), but this exercise focuses on using familiar techniques (primarily electrophoresis) to cross the threshold of a new field, proteomics.