Immunoblotting Immediately after completing

the electrophoresis run, OMPs and LPS were transferred to nitrocellulose (NC) membranes according to Harlow and Lane [28] with some modifications. Gels and NC membranes were soaked in Tris-glycine transfer buffer (10% [v/v] methanol, 24 mM Tris base, 194 mM glycine) for 15 min. Separated OMPs and LPSs were transferred onto NC using a mini-transblot cell (Bio-Rad). The membranes were blocked with 3% (w/v) Selumetinib in vivo BSA in Tris Buffered Saline (TBS) containing Tween 20 (0.05% v/v). NC membranes were then incubated with affinity purified MAbs (2 μg ml-1) diluted in 0.15 M TBS buffer containing 1% (w/v) BSA with gentle shaking for 1 h. Membranes were then developed with goat anti-mouse-HRP in 0.15 M TBS buffer containing 1% (w/v) BSA and a diaminobenzidine (DAB) substrate solution. Color development

was stopped by rinsing the membranes with distilled water. Protein sequencing and identification Extracted OMPs were separated on SDS-PAGE gels and probed with anti-OMP monoclonal antibodies. Immunoblot-positive bands were cut with sterile sharp scalpel and immersed in 1% acetic acid solution. Protein sequencing was performed using the MALDI-TOF technology at the Proteomics and Mass Spectrometry Facility at Purdue University (West AP24534 concentration Lafayette, Indiana, USA). Dot blot assay Dot blotting was performed as described by Jaradat and Zawistowski [23]. One microliter of heat-killed Cronobacter whole-cell suspension (108 cells ml-1) was Selleckchem CP673451 spotted on the NC membranes, allowed to air dry for 30 min and incubated in 5% (w/v) NaOH or in 38% (v/v) HCl for 10 s or left untreated. Immunoblotting was performed as described above. Immunoelectron microscopy Immunolabeling was performed essentially as described by Jaradat and Zawistowski [23] with modifications. Briefly, 5 μl of bacterial suspension in distilled water (5 × 108 CFU ml-1) were placed on formvar-coated copper grids. After air-drying for 2 h at room temperature,

Ketotifen the grids were blocked with PBS containing 3% (w/v) BSA for 30 min at 37°C. To expose antigens on bacteria, grids were incubated with 0.1 M NaOH or 0.1 M HCl for 2 h, washed with water and incubated with purified MAb solution at 37°C. Grids were then incubated with colloidal gold (18 nm)-conjugate anti-mouse IgG diluted at 1:50 in dilution buffer (0.02 M Tris, 150 mM NaCl, 0.1% [w/v] BSA, 0.005% [v/v] Tween 20, 0.4% [w/v] gelatin [pH 9]) for 20 h at room temperature. Grids were washed 6 times with water and viewed with a Zeiss Transmission Electron Microscope at various magnifications. Animal use Animals used for immunization and production of monoclonal antibodies were cared for according to the Animal Care and Use Committee (ACUC), Jordan University of Science and Technology. Results Two approaches were attempted to produce monoclonal antibodies specific to Cronobacter spp.: one group of mice was immunized with heat-killed C.