Purification of recombinant His-tagged proteins and preparation of polyclonal antisera PIII and NG1873 His-tagged proteins were expressed in E. coli as described [25] and proteins were purified on a metal-chelate affinity chromatography column (MCAC); proteins were eluted in a single step with 50 mM phosphate buffer pH 8.0, 300 mM NaCl, 250 mM imidazole and protease inhibitors. To prepare antisera against PIII and NG1873 His-tagged proteins, learn more 20 μg of each purified protein were used to immunize CD1 female mice. The recombinant proteins were given i.p. together with Al(OH)3 for three doses (day 0, day 21, day 35). Blood samples were taken on day 49 and pooled. Confocal immunofluorescence

microscopy To visualize PIII protein on bacterial selleck products surface, F62 strain was grown in GC up to OD600 0.5 and washed in PBS. Bacterial pellet were fixed with 2% PFA for 20 min at room temperature and spotted on chamber slides coated with poly-lysine. Bacteria were then blocked with 2% BSA for 15 min

and incubated with mouse polyclonal anti-PIII antibodies diluted in 2% BSA for 30 min at room temperature. Bacteria were then stained with goat anti-mouse Alexa Fluor 568 conjugated antibodies (Molecular Probes) for 20 min at room temperature. Labeled samples were mounted with ProLong® Gold antifade reagent with DAPI and analyzed with Zeiss LSM710 Ilomastat chemical structure confocal microscope. Negative staining and TEM A drop of a 109 cfu/mL bacterial suspension in D-PBS was placed on Parafilm and bacteria were

adsorbed for 15 min to formvar/carbon 200 mesh grids. Bacteria were fixed for 15 min with 2% p-formaldehyde and grids were then rinsed four times in PBS and air-dried. Grids were finally treated with uranyl acetate and examined by TEM GEOL 1200EX II transmission electron microscope. Paper disk diffusion inhibiting assays F62 and F62ΔpIII strains were grown overnight on GC agar, suspended in D-PBS and adjusted to OD600 = 0.1 (≅108 cfu/mL). An aliquot of 0.1 mL of the bacterial suspension was seeded on GC agar. 10 μL of the following detergents 17-DMAG (Alvespimycin) HCl were applied to paper disk (Oxoid): SDS at 0.125, 0.25, 0.5, 1%, Triton X-100 at 0.03, 0.06, 0.125, 0.25% and deoxycolate at 0.8, 0.9, 1.2, 1.4%. Control disks with PBS were included in the assay. Disks were then placed on the GC agar inoculated with bacteria. All plates were incubated at 37°C in 5% CO2. Cell fractionation and protein analysis Total cell lysates (TL), inner membranes (IM) and outer membrane (OM) were prepared from bacteria at exponential growth phase. Total cell lysates were obtained by three freezing-thawing cycles. For IM and OM preparation, bacteria were sonicated, unbroken cells were removed by centrifugation and the supernatant centrifuged at 50000 × g for 90 min at 4°C. The pellet containing the membranes was incubated in 2% Sarkosyl in 20 mM Tris–HCl, pH 7.5 at room tempertaure for 20 min to solubilize the inner membranes.