PREPARATIVE ISOELECTRIC-FOCUSING AND PREPARATIVE ELECTROPHORESIS OF HYDROPHOBIC CANDIDA-ALBICANS CELL-WALL PROTEINS WITH IN-LINE TRANSFER TO POLYVINYLIDENE DIFLUORIDE MEMBRANES FOR SEQUENCING

Hydrophobic proteins in the cell wall of the opportunistic fungal path ogen Candida albicans are involved in adhesion of this organism to hos t tissue and thus play a role in its pathogenicity. The hydrophobic na ture of these proteins results in their loss during purification due t o adsorption to apparatus surfaces. This problem, combined with their low abundance, has made it problematic to purify the hydrophobic prote ins in sufficient quantity for sequencing or biochemical analysis. We describe a system that combines preparative isoelectric focusing with continuous elution preparative electrophoresis. The system provides a two-dimensional protein separation while maintaining protein solubilit y and minimizing protein loss due to adsorption. In addition, we have added an in-line transfer of electrophoretic fractions directly to pol yvinylidene difluoride (PVDF) membranes, which further reduces both ex posure to apparatus surfaces and purification time.