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Preparative isoelectric focusing and preparative electrophoresis of hydrophobic Candida albicans cell wall proteins with in‐line transfer to polyvinylidene difluoride membranes for sequencing
Author(s) -
Masuoka James,
Glee Pati M.,
Hazen Kevin C.
Publication year - 1998
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150190512
Subject(s) - isoelectric point , chromatography , electrophoresis , membrane , isoelectric focusing , chemistry , elution , adsorption , gel electrophoresis , two dimensional gel electrophoresis , biochemistry , proteomics , organic chemistry , enzyme , gene
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.