After each wash the bacteria were pelleted by centrifugation. Finally, the Streptococcal pellet was re-suspended in PBS containing 2% (w/v) SDS, vortexed and incubated at room temperature for 1 h. Next, the

SDS-extract was centrifuged at 10,000 rpm at 4°C for 10 min and the supernatant containing surface extract was stored at -80°C for further use. Protein content of the extracts was measured by BCA protein assay kit (Pierce Chester, UK). Analytical SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was selleck chemical performed in a LKB 2050 mini-gel electrophoresis unit in a discontinuous gel system under non-reducing conditions. Samples were mixed with loading buffer [1.25 M Tris-HCl/10% SDS (w/v)/50% (v/v) glycerol containing 0.02% (w/v) bromophenol blue]. Gels were Selleckchem Rigosertib electrophoresed (running buffer; 0.2 M Glycine/0.25 M Tris-HCl, pH 8.3 containing 0.1% (w/v) SDS) at 120 V until the dye front reached the end of

the gel. Prestained broad range molecular weight markers were run on every gel. Following Veliparib electrophoresis, gels were stained with Brilliant blue G-colloid for 2 h, then destained with repeated rinses of 25% (v/v) methanol. Molecular masses of the proteins were automatically calculated in a Bio-rad model GS-700 imaging densitometer with the Profile analyst II, V. 3.11 software. Preparative SDS-PAGE The streptococcal cell surface extract was fractionated on a Bio-Rad Model 491 Prep Cell. A 5 ml sample containing 20 mg Streptococcal surface protein was loaded on a mini-Prep

Cell tube (diameter of 37 mm) prepared with a 9 cm 7.5% separating and 4 cm 4% stacking gel. The sample was electrophoresed at Histone demethylase 4°C, at constant 60 mA and the elution buffer (0.2 M Glycine/0.25 M Tris-HCl, pH 8.3 containing 0.1% (w/v) SDS) flow velocity of 125 μl/min. 2.5 ml fractions were collected and stored at -80°C for further use. Western transfer of SDS-PAGE gels Gels were equilibrated in transfer buffer [250 mM Tris/20% (v/v) methanol/200 mM glycine containing 0.1% (w/v) SDS] for 15 min prior to transfer to 0.2 μm pore size nitrocellulose membranes using semidry electrotransfer with a Pharmacia-LKB Multiphore II Novablot unit. Transfer conditions were 60 mA constant for 1 h. Identical blots were stained with amido black (0.2% (w/v), containing 3% (w/v) TCA) and destained with methanol, to check transfer efficiency. For enolase immunoblotting, the membrane was probed with an antibody raised against human enolase (C-19, Santa Cruz) which was shown to cross-react with streptococcal enolase [34]. Immuno-detection was performed using ECL detection. Blot overlay assay to detect MUC7-binding proteins from S. gordonii MUC7-binding proteins were determined by an immunoblotting procedure using the monoclonal antibody AM-3. This antibody is reactive against the oligosaccharide structure sialyl-Lewisx which is present on MUC7 [35, 36].