UNIT 22.6 Protein Profiling by Microscale Solution Isoelectrofocusing (MicroSol-IEF)

Abstract

Sample prefractionation is essential for more comprehensive coverage and reliable detection of low-abundance proteins in complex proteomes. An efficient and reproducible new method for sample prefractionation is microscale solution isoelectrofocusing (MicroSol-IEF), in which samples are separated into chambers defined by membranes of specific pH, yielding well resolved fractions on the basis of isoelectric point (pI). The output seamlessly interfaces with narrow-pH-range 2-D gels, enhancing data obtained from protein profiling studies, including quantitative proteome comparisons. This unit presents the MicroSol-IEF method using the ZOOM IEF Fractionator with either commercially available or custom-made pH partition membranes. Alternative configurations are possible for separating samples into different numbers of fractions with various pH ranges and volumes. A detailed method is provided for preparing custom pH membranes. In addition, methods are provided for evaluating the effectiveness of the prefractionation, using 1-D and 2-D gel electrophoresis. Approaches for quantitative protein profiling that incorporate MicroSol-IEF are also discussed.