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 pathogen Candida albicans are involved in adhesion of this organism to host tissue and thus play a role in its pathogenicity. The hydrophobic nature of these proteins results in their loss during purification due to adsorption to apparatus surfaces. This problem, combined with their low abundance, has made it problematic to purify the hydrophobic proteins 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 solubility and minimizing protein loss due to adsorption. In addition, we have added an in-line transfer of electrophoretic fractions directly to polyvinylidene difluoride (PVDF) membranes, which further reduces both exposure to apparatus surfaces and purification time.
Masuoka
J
J
Department of Pathology, University of Virginia Medical Center, Charlottesville 22908, USA. jm2n@Virginia-edu
Glee
P M
PM
Hazen
K C
KC
D015153:Blotting, Western; D002176:Candida albicans / Q000737:chemistry / Q000235:genetics*; D002473:Cell Wall / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics*; D007525:Isoelectric Focusing / Q000379:methods*; D013058:Mass Spectrometry; D008567:Membranes, Artificial; D011145:Polyvinyls; D017421:Sequence Analysis; D005465:Fluorocarbon Polymers
D005656:Fungal Proteins; D008567:Membranes, Artificial; D011145:Polyvinyls; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers