Solubilization of cellular membrane proteins from Streptococcus mutans for two-dimensional gel electrophoresis.
Electrophoresis
; 26(6): 1200-5, 2005 Mar.
Article
em En
| MEDLINE
| ID: mdl-15706571
ABSTRACT
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.
Buscar no Google
Base de dados:
MEDLINE
Assunto principal:
Streptococcus mutans
/
Eletroforese em Gel Bidimensional
/
Proteínas de Membrana
Idioma:
En
Ano de publicação:
2005
Tipo de documento:
Article