Cell surface proteins of Candida albicans: preparation of extracts and improved detection of proteins.
We have reexamined the detection of the components in a beta-mercaptoethanol and ammonium carbonate buffer extract of surface proteins of Candida albicans and the effects of postextraction manipulation of the extract on recovery of extract components. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), preferential staining of some moieties was observed when bands detected by a commercial silver staining method or a Coomassie Brilliant Blue (CBB) staining method were compared. Additional protein bands that were either not detected or poorly detected by a single method alone were readily observed by a combined silver-CBB staining method. This method also detected alterations in the profile of extracted proteins from organisms grown in the presence of galactose or hemoglobin rather than glucose. Two-dimensional electrophoresis (2-DE) gel analysis by double stain showed better detection of several acidic and basic protein spots. Less than 10% of the extract as determined by a dye-binding assay was lost following either or both lyophilization and dialysis. These manipulations of the extract did not change the protein profile following SDS-PAGE as determined by the combined staining or Western blot analysis of a 70 kDa protein. These observations suggest that soluble cell wall proteins are not unusually sensitive to procedures routinely used in protein purification. In addition, these studies suggest that a modified staining method that combines both silver stain and CBB stain provides improved detection of cell wall proteins compared to either method alone.
Vediyappan
G
G
Department of Microbiology and Immunology, Texas Tech University Health Sciences Center, Lubbock 79430, USA.
Bikandi
J
J
Braley
R
R
Chaffin
W L
WL
D001557:Benzenesulfonates; D015153:Blotting, Western; D002021:Buffers; D002176:Candida albicans / Q000737:chemistry* / Q000254:growth & development; D002254:Carbonates; D002473:Cell Wall / Q000737:chemistry*; D004396:Coloring Agents; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000032:analysis*; D005690:Galactose / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000032:analysis*; D008623:Mercaptoethanol; D016622:Silver Staining; D013194:Staining and Labeling
D001557:Benzenesulfonates; D002021:Buffers; D002254:Carbonates; D004396:Coloring Agents; D005656:Fungal Proteins; D008565:Membrane Proteins; D008623:Mercaptoethanol; C006796:brilliant blue; D005947:Glucose; C040502:ammonium carbonate; D005690:Galactose