Preparation of Mammalian Tissue Samples for Two-Dimensional Electrophoresis

Abstract

The true power of two-dimensional gel electrophoresis (2-DE) requires the careful preparation of protein samples to minimize sample variability and maximize solubilization. This is of particular relevance when 2-DE is relied upon to characterize the differential expression of mammalian tissue proteomes in large experiments with large numbers of samples. One of the weaknesses of the 2-DE approach relates to its depth of field-that is, its limited ability to resolve reproducibly those proteins with extreme isoelectric points (pI) (e.g., >3 and <9), hydrophobicity, and low abundance. Second, the initial step in 2-DE of protein mixtures, isoelectric focusing, is susceptible to a number of problems that cause variability in the final protein pattern, interferences that must be avoided. Hence, a simple yet reproducible method of preparing cell and tissue samples for consistent 2-DE results, involving as little manipulation as possible, is of utmost importance.