Structural characterization of blotting membranes and the influence of membrane parameters for electroblotting and subsequent amino acid sequence analysis of proteins.

Various blotting membranes were evaluated and correlated with the efficiency of electroblotting and the performance in the sequencing process. Structural parameters including specific surface area, pore size distribution, pore volumes, and permeabilities of different solvents lead to discrimination of the membranes relative to their accessible surfaces and membrane densities. Protein binding capacities as well as protein recoveries in electroblotting correlate with the specific surface areas. Almost quantitative retention of proteins during electroblotting from gels was obtained for membranes with a high specific surface area and narrow pores (Trans-Blot, Immobilon PSQ, Fluorotrans), whereas membranes with a relatively low specific surface area (Immobilon P, Glassybond) showed reduced recoveries of between 10-20% for the tested proteins. Initial yields and repetitive yields were compared for radioiodinated standard proteins that have been either electroblotted or loaded by direct adsorption. The results showed that the different permeabilities for solutions of the Edman chemistry have a major influence on initial yields. The glass fiber-based membranes with an extremely low flow restriction produce consistently high initial yields independent of the application mode of the protein (spotted or electroblotted) or the application of the membranes into the cartridge (discs or small pieces). In contrast, the polymeric membranes showed decreasing initial yields with increasing membrane density for spotted and electroblotted proteins. Yields varied considerably when the membranes were applied as discs into the cartridge. This effect could be minimized by cutting the membranes into pieces as small as possible, as demonstrated for electroblotted proteins.