Representative strains of the three previously described groups of V. tapetis

(Rodríguez et al., 2006) with different phenotypical, serological and genetic profiles as well as different host origin were used in this study: CECT 4600T, type strain of the species isolated from Manila clam (Ruditapes philippinarum), GR0202RDRD obtained from carpet shell clam (Ruditapes decussatus) and HH6087 isolated from halibut (Hipoglossus hipoglossus) ABT-263 nmr (Borrego et al., 1996; Novoa et al., 1998; Reid et al., 2003). The bacteria were routinely grown aerobically on marine agar (MA) (Pronadisa, Spain) at 15 °C for 72 h. Stock cultures were maintained frozen at −80 °C in marine broth (MB) (Pronadisa) supplemented with 15% glycerol (v/v). Bacterial inocula with 109 cells mL−1 were prepared by diluting the bacterial suspension to an OD of 1 (OD580 nm). For each strain, 1 L of sterile MB was inoculated

to achieve a final concentration of 105 cells mL−1 and was aerobically incubated in a Innova 4340 rotary shaker (70 r.p.m.) (New Brunswick Scientific) at 15 °C for 72 h. Bacteria were harvested and washed with Tris–buffered sucrose http://www.selleckchem.com/products/INCB18424.html (10 mmol Tris, 250 mmol sucrose pH 7) and lyophilized. Proteins were extracted by suspending 40 mg of lyophilized bacteria in 1 mL standard lysis buffer – 7 M urea, 2 M thiourea, 4% CHAPS [3-(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate] – and 65 mM dithiothreitol (DTT) for 3 h at 27 °C and sonication (three cycles of 10 pulses). Next,

samples were centrifuged at 12 000 g for 30 min and supernatants were collected and subjected to protein precipitation using the Clean-up kit (GE Healthcare, Sweden). After suspension of the pellet in 1 mL of lysis buffer, the protein concentration was measured with a CB-X protein assay kit (Gbiosciences). Finally, samples were stored at −80 °C prior to use. Isoelectrofocusing (IEF) was performed using a Protean IEF cell (Bio-Rad) and 24 cm pH 4-7 IPG strips (GE Healthcare). Liothyronine Sodium For each sample, 400 μg of protein was resuspended in 390 μL of rehydration buffer (7 M urea, 2 M thiuorea, 4% CHAPS, 0.6% DTT, 1% IPG buffer 4-7 and bromophenol blue traces). IEF was carried out at 20 °C in the following steps: active rehydration (50 V) for 12 h, 250 V for 30 min, 500 V for 1 h, 1000 V for 1 h, 4000 V for 2 h, 8000 V for 2 h and 10 000 V, to achieve 65 kVh. Prior to running the second dimension, strips were equilibrated at room temperature for 15 min with an equilibration solution [6 M urea, 50 mM Tris–HCl pH 8.8, 30% glycerol, 2% sodium dodecyl sulfate (SDS)] with the addition of 1% DTT, and for other 15 min in the same solution supplemented with 2.5% iodoacetamide. Strips were placed on top of a 21 ×26 cm 12.5% polyacrylamide gel and fixed with sealing solution (25 mM Tris, 192 mM glycine, 0.1% SDS, 0.5% agarose, 0.01% bromophenol blue).