A novel, Bicine-based SDS-PAGE buffer system was developed for the analysis of membrane proteins. The method involves molecular weight-based separations of fully denatured and solubilized proteins in two dimensions. This doubled SDS-PAGE (dSDS-PAGE) approach produced a diagonal arrangement of protein spots and successfully circumvented problems associated with membrane proteome analysis involving traditional gel-based methods. Membrane proteins from the anaerobic bacterium Clostridium thermocellum were used for these investigations. Tricine-dSDS-PAGE and the newly developed Bicine-dSDS-PAGE were compared with the standard glycine-dSDS-PAGE (Laemmli protocol) in their suitability to separate C. thermocellum membrane proteins. Large-format gel experiments using optimized gel preparation and running buffer conditions revealed a 112% increase in protein spot count for Tricine-dSDS-PAGE and a 151% increase for Bicine-dSDS-PAGE, compared to glycine-dSDS-PAGE. The data clearly indicated that Bicine-dSDS-PAGE is a superior method for the analysis of membrane proteins, providing enhanced resolution and protein representation.