Preparative isoelectric focusing and preparative electrophoresis of hydrophobic Candida albicans cell wall proteins with in‐line transfer to polyvinylidene difluoride membranes for sequencing

Abstract

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.

Analysis of intact proteins from cerebrospinal fluid by matrix-assisted laser desorption/ionization mass spectrometry after two-dimensional liquid-phase electrophoresis

“…27 Because mass spectrometric analysis of proteins blotted onto membranes has limited sensitivity and resolution, we preferred our combination of methods. One of the most important advantages of 2D-LPE is that the proteins still remain in solution throughout the analysis and extra steps such as electroblotting or extraction from gels are avoided.…”

Isoelectric focusing nonporous silica reversed‐phase high‐performance liquid chromatography/electrospray ionization time‐of‐flight mass spectrometry: a three‐dimensional liquid‐phase protein separation method as applied to the human erythroleukemia cell‐line

“…Other groups have used multi‐dimensional liquid‐phase separations to prepare proteins for analysis by MALDI and/or ESI mass spectrometry 16. This kind of work has included 2‐D liquid chromatography,17,, 18 1‐D liquid‐phase IEF,16,, 19,, 20 1‐D continuous elution PAGE,21 2‐D preparative LPE,19,, 20,, 22 2‐D semi‐preparative electrophoresis,16,, 20,, 23 and IEF‐RP‐HPLC 24…”

Two Dimensional Gel Electrophoresis in Cancer Proteomics

“…Techniques such as differential extraction , subcellular fractionation (Taylor et al 2000, Morel et al 2000, chromatography , or prefocusing in a preparative IEF device such as the Rotofor® system (Masuoka et al 1998, Nilsson et al 2000 have been used to reduce the complexity of samples.…”