Solubilization of cellular membrane proteins fromStreptococcus mutans for two-dimensional gel electrophoresis
| Autor: | Kheir Zuobi-Hasona, Paula J. Crowley, L. Jeannine Brady, Adnan Hasona, Arnold S. Bleiweis |
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| Rok vydání: | 2005 |
| Předmět: |
Two-dimensional gel electrophoresis
Chromatography Octoxynol Detergents Clinical Biochemistry Aqueous two-phase system Membrane Proteins Sodium Dodecyl Sulfate Trifluoroethanol Biochemistry Polyethylene Glycols Analytical Chemistry Streptococcus mutans Electrophoresis Chaotropic agent chemistry.chemical_compound Membrane Solubility chemistry Membrane protein Electrophoresis Gel Two-Dimensional Chloroform Sodium dodecyl sulfate Hydrophobic and Hydrophilic Interactions |
| Zdroj: | ELECTROPHORESIS. 26:1200-1205 |
| ISSN: | 1522-2683 0173-0835 |
| DOI: | 10.1002/elps.200410349 |
| Popis: | Membrane proteins are rarely identified in two-dimensional electrophoretic (2-DE) proteomics maps. This is due to low abundancy, poor solubility, and inherent hydrophobicity leading to self-aggregation during the first dimension. In this study, membrane proteins from the Gram-positive bacterium Streptococcus mutans were solubilized using three different methods and evaluated by 2-DE. In the first method, the extraction was performed using sodium dodecyl sulfate (SDS) followed by solubilization with a chaotropic buffer and precipitation with methanol/chloroform. The second method was based on temperature-dependent phase partitioning using Triton X-114 followed by purification using the ReadyPrep 2-D clean-up kit from Bio-Rad. The third method involved extraction using the organic solvents trifluoroethanol (TFE) and chloroform, which produced three separate phases. The upper aqueous phase, enriched with TFE, gave the highest overall protein yield and best 2-DE resolution. Protein spot identification by nanoelectrospray quadrupole time of flight (QTOF)-tandem mass spectrometry (MS/MS) revealed known membrane and surface-associated proteins. This is the first report describing the successful solubilization and 2-D electrophoresis of membrane proteins from a Gram-positive bacterium. |
| Databáze: | OpenAIRE |
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