A Combined Free Flow Electrophoresis and DIGE Approach to Compare Proteins in Complex Biological Samples

Abstract

Free flow electrophoresis (FFE) has been applied in numerous studies as a protein separation technique due to its multiple advantages such as fast and efficient sample recovery, high resolving power, high reproducibility, and wide applicability to protein classes. As a stand-alone platform however, its utility in comparative proteomic analysis is limited as protein samples must be run sequentially rather than simultaneously which introduces inherent variability when attempting to perform quantitative analysis. Here we describe an approach combining fluorescent CyDye technology (DIGE) with FFE to simultaneously separate and identify differentially expressed proteins in a model cell system.

Sodium dodecyl sulfate free gel electrophoresis/electroelution sorting for peptide fractionation

“…In its most successful application, IEF is used as a first dimension of two dimensional electrophoretic systems, as reported since 1975 . For shotgun proteomics, protein separation by IEF has been used as a pre‐fractionation step, previous to the LC–MS/MS analysis . For this aim, several instrument designs based on IEF fundamentals have been developed.…”