Strips were focused on an IPGphor (GE Healthcare) for a total of 60,000 Vh. After focusing, strips

were equilibrated in 50 mM EPZ 6438 Tris-HCl, pH 6.8, 2% SDS, 7 M urea, 10% glycerol, supplemented with 2% DTT for 15 min, and then with 2.5% iodoacetamide for 15 min. The second dimension (SDS-PAGE) was conducted on 10% to 18% polyacrylamide see more gradient gels, on an Ettan DALTsix electrophoresis system (GE Healthcare), following manufacturer’s instructions. 2-D gels were silver stained with a mass-compatible method [53] and images were digitalized with an Image Scanner (GE Healthcare). 2D DIGE For 2D DIGE analysis, the two field isolates Bortigali and Nurri were compared to PG2T. Triton X-114 Protein extracts were precipitated and resuspended in lysis buffer as described above. Then, samples were labeled with CyDye DIGE Fluors (GE Healthcare)

according to the minimal labeling protocol provided by the manufacturer. Briefly, after CyDye reconstitution with dimethylformamide (DMF) and preparation of a working solution (200 pmol/μL), 1 μL of diluted CyDye was added to a volume of protein sample equivalent to 50 μg. Samples were left on ice for 30 minutes in the dark, and then 1 μL of 10 mM lysine was added to stop the reaction. Labeled samples were mixed, IPG buffer corresponding to the desired pH range was added at a 1% final concentration, and DeStreak Rehydration Solution (GE Healthcare) was added to a total volume of 340 selleck chemicals llc μL. 18 cm IPG strips (GE Healthcare) were passively rehydrated for at least 6 hours. IEF was carried out on an Ettan IPGphor II (GE Healthcare) for a total of ~60,000 Vh. After focusing, strips were equilibrated in 50 mM Tris-HCl, pH 6.8, 2% SDS, 7 M urea, 10% glycerol, supplemented with 2% DTT for 10 min, and then with 2.5% iodoacetamide for 10 min. Proteins were then subjected to SDS-PAGE

in 10-18% gradient polyacrylamide gels on the Ettan DALTsix system (GE Healthcare), following the manufacturer instructions. DIGE images were detected with a Typhoon Scanner (GE Healthcare) and processed with DeCyder (GE Healthcare) for image analysis. Western Immunoblotting SDS-PAGE resolved proteins were transferred onto nitrocellulose membranes with a Mini-Trans-Blot Cell (Bio-Rad) Tangeritin at 250 mA for one hour. After blotting, membranes were blocked with PBS-0.05% Tween 20 (PBS-T) containing 3% BSA. Membranes were incubated for one hour with a rabbit hyperimmune serum raised against M. agalactiae recombinant P48 (M. agalactiae rP48) [12]. Membranes were washed five times with PBS-T and incubated with the appropriate HRP-conjugated secondary antibodies (Sigma). After five washes, membranes were developed with SuperSignal West Pico Chemiluminescent Substrate (Pierce) and images were acquired with a VersaDoc MP 4000 Imaging System (Bio-Rad).