Proteinase-K
digested H. pylori The procedure selleck compound was followed as described previously [30]. H. pylori cells were collected and adjusted to a concentration of 2.5 × 109 cells/ml in PBS. Bacteria were boiled with 150 μl sample dye for 10 min at 100°C to disrupt the whole cells. Subsequently, the whole cell lysates were treated with proteinase K (Sigma) for 60 min at 60°C in a water bath. Then, 2.5 × 108 cells/ml were analyzed by 12% SDS-PAGE and stained with silver. The protein concentration of the 2.5 × 108 cells/ml was also determined by using the Bio-Rad protein assay (Bio-Rad) to serve as a loading control. Immunoblots of LPS from H. pylori with anti-Lewis (Le) monoclonal antibody H. pylori strains that have been screened serologically [31–33], www.selleckchem.com/products/z-vad-fmk.html and a previous study suggested that Asian isolates express predominantly type 1 (Lea, Leb) antigens compared to Western strains (predominantly expressing type 2 Lex and Ley determinants) [34]. We also primarily detected the Lewis antigen of NTUH-S1 with anti-Lea and anti-Leb antibody. Equivalent amounts of protein were loaded in each well and transferred to nitrocellulose for immunological detection with anti-Lea or anti-Leb monoclonal
antibody (Seikagaku Corporation, Tokyo). For detection of Lewis antigen in proteinase K-digested whole cell lysates, nitrocellulose membrane was blocked with 5% skimmed milk in PBS for 1 h at room temperature. Subsequently, membrane was incubated with anti-Lea or anti-Leb antibody diluted 1:3000 with 5% skimmed milk in PBS overnight at 4°C. Horseradish peroxidase-conjugated anti-mouse IgG diluted 1:5000 with 5% skimmed milk in PBS was added and membrane was incubated
for 1 h at room temperature. The membrane was washed three times with PBST (0.05% Tween-20) between the incubation steps. Electrochemiluminescence (Amersham Biosciences) was used for detection. Whole cells of the Lex and Ley antigen-expressing H. pylori 26695 strain [35] were used as a negative control in Western blots to ensure the specificity of the anti-Lea or anti-Leb antibody. Measurement of outer membrane permeability by ethidium bromide Outer membrane permeability can be measured Ureohydrolase by the fluorescence of the ethidium-polynucleotide complex in the cell because ethidium bromide displays approximately a 10-fold increase in fluorescence quantum yield upon binding to DNA [36]. The assay was modified as described previously [37]. Briefly,H. pylori were grown on Columbia blood agar plate for 48 h. Then, bacteria were pelleted and washed twice with ice-cold 50 mM potassium phosphate (pH 7.0) containing 5 mM MgSO4. Cells were resuspended in 1 ml of potassium phosphate buffer (pH 7.0) at an optical density (OD600) of 0.5 and incubated for 30 min at 37°C in the presence of 10 μM of CCCP to deplete cells of metabolic energy. Subsequently, cells were washed three times in ice-cold potassium phosphate (pH 7.0) containing 5 mM MgSO4 and loaded with 10 μg/ml ethidium bromide.