0 containing 0.05% (v/v) Tween 20) supplemented with 1% (w/v) skimmed milk powder 30 min. They were rinsed twice with PBS 10 min, and GSK2126458 manufacturer incubated with MUC7 preparation (10 μg/ml in PBS) at 4°C overnight. In the meantime, a replica membrane was incubated with PBS as control. After the incubation the

membranes were rinsed twice for 20 min with TBST. The membranes including replica control, were then incubated with AM-3 in TBST (1:50 dilution) for 1 h, then rinsed Selumetinib ic50 with TBST 2 × 10 min and incubated with secondary antibody (IgM anti-mouse, peroxidase conjugated, 1:2000 dilution) in TBST for 30 min. The membranes were rinsed with TBST 3 × 10 min. ECL detection was carried out using an Amersham ECL kit according to the manufacturer’s instructions. Anti-enolase labelling

and flow cytometry analysis of the bacteria S. gordonii suspension was adjusted to OD at 250 nm of 0.5 with PBS and incubated with an anti-enolase antibody (C-19, Santa Cruz) overnight at 4°C with end-over-end rotation. The bacteria were harvested by centrifugation at 3000 × g at 4°C, washed twice with ice-cold PBS. Texas Red-labeled anti-goat IgG (Jackson ImmunoResearch) secondary antibody was added to the bacterial suspension and incubated for 30 min and then washed with PBS as described above. Purified goat IgG (Invitrogen) was incubated with the bacteria and used as isotype-matched control. Samples were analyzed by a CyAn ADP flow cytometer (Beckman Coulter) and the data were analyzed using Summit software selleck products version 4.3. A minimum of 2 × 104 Bumetanide cells per sample were examined. In-gel digestion A previously described method [37] was used for in-gel digestion of the putative adhesins with some minor modifications. Briefly, the protein band was cut out from the SDS-PAGE gel and transferred into a 1.5 ml eppendorf tube; all subsequent steps were performed in the same tube. Gel pieces were de-stained with 50 mM NH4HCO3 in 50% acetonitrile and then reduced with 10 mM dithiothreitol in 50 mM NH4HCO3 at 37°C for 1 h prior to alkylation by addition of 55 mM iodoacetamide 1 h in the dark at room

temperature. The gel pieces were washed in 100 mM NH4HCO3 before dehydrating in acetonitrile and then rehydrating in 100 mM NH4HCO3. Gel pieces were dehydrated once again in acetonitrile and dried in the vacuum centrifuge (about 30 min). Trypsin (1 ng/μl in 50 mM NH4HCO3) was added to the dried gel pieces and left for 30 min in ice. Excess digestion buffer was replaced with the same buffer (10 μL) without trypsin and the gel pieces were incubated 24 h at 37°C. Extraction of the peptides was performed in two steps; 50 μL of 25 mM NH4HCO3 for 30 min and 50 μL of 5% (v/v) formic acid in 50% acetonitrile (v/v) 2 × 20 min. Extracts obtained from each step, were combined, then dried down and analyzed by LC MS/MS.