The Use of Two-Dimensional Gel Electrophoresis for Plasma Biomarker Discovery

Two-dimensional polyacrylamide gel electrophoresis (2-DE) is one of the most widely used and versatile methods of protein expression profiling among a rapidly growing arsenal of proteomics technologies. 2-DE combines two orthogonal and independent electrophoretic steps: isoelectric focusing (IEF) and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). At present, 2-DE is capable of simultaneously detecting and quantifying up to several thousand protein spots in the same gel image. As an example to illustrate the power of 2-DE, we provide comprehensive step-by-step instructions for the application of 2-DE to clinically collected human plasma samples to determine whether specific plasma proteins or protein expression patterns could serve as biomarkers for the development of a specific adverse side effect that arises upon treatment with an experimental drug. These instructions provide detailed information on how to deplete high-abundant proteins from human plasma, apply the depleted plasma proteins to immobilized pH gradient gels, perform IEF and SDS-PAGE separation, and perform silver staining to visualize proteins. The basic 2-DE protocol described here could easily be applied to other tissue types.