Strips were rinsed briefly with 25% 1.5 M pH 8.0 Tris before SDS–PAGE was performed using Criterion 12.5% Tris-HCl Precast gels (Bio-Rad), run at 200 V for approximately 45 min. For each sample, two gels were run simultaneously, one for silver staining and another for
immunoblotting. Gels for silver staining were fixed individually in 0.1 L fixing solution [50% (v/v) methanol, 10% (v/v) acetic acid] for find more a minimum of 1 h, and were subsequently stained using a sensitive ammoniacal silver method based on silver nitrate. Gel images were acquired using the UMAX Powerlook 1000 flat-bed scanner. Proteins from unstained gels were transferred electrophoretically onto polyvinylidene fluoride (PVDF) membranes using the Trans-blot cell transfer system (Bio-Rad Laboratories). Tigecycline cost To visualize total proteins, membranes were stained with a Sypro Ruby blot stain (Bio-Rad
Laboratories). To detect immunoreactive proteins, membranes were destained and subsequently probed according to the Immun-Star™ WesternC™ kit protocol (Bio-Rad Laboratories). Membranes were immunolabeled with patients’ sera at a 1 : 250 dilution, and goat anti-human IgG antibodies coupled to HRP (1 : 2000; Bio-Rad) were used as a secondary antibody. The immunoreactive protein spots matched using both the Sypro Ruby stained membrane and the silver-stained gels were identified by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Briefly, spots were washed twice for 10 min in 200 μL of 100 mM NH4HCO3, reduced at 37 °C for 1 h with 50 μL of 10 mM DTT, alkylated for 1 h in 50 μL of 10 mM iodoacetamide, washed for 10 min with 200 μL of 10 mM NH4HCO3, dehydrated in acetonitrile, and trypsin-digested with 10 ng μL−1 of trypsin (Promega, Annandale, NSW, Australia). After digestion for 14 h at 37 °C, peptides were extracted by washing the gel slice for 15 min with 25 μL 1% formic acid, followed by dehydration in acetonitrile. Digests were then dried in vacuo, resuspended in 10 μL 1% formic acid and submitted for
a Quadrupole-TOF analysis on a Micromass instrument which generated collision-induced dissociation. Results were analyzed using the Mascot MS/MS ion search (Matrix Science, Boston, MA), and searches were performed on the National Centre for Biotechnology Phosphoglycerate kinase Information non-redundant (NCBI nr) database (specifically against the available genome sequence of C. concisus BAA-1457). This study was approved by the Research Ethics Committees of the University of New South Wales and the South East Sydney Area Health Service-Eastern Section, Sydney (Ethics No.: 03/163, 03/165 and 06/164). Recently, an association between the presence of C. concisus DNA and newly diagnosed CD was reported in two case–control studies using intestinal biopsies and fecal samples (Zhang et al., 2009; Man et al., 2010c). In addition, significantly higher levels of C. concisus-specific IgG antibodies were detected in children with CD as compared with controls (Zhang et al.