5% SDS, 10 mM Tris; pH 6.9) followed by incubation at 37°C for 30 min. After centrifugation (16,100 × g for 10 min at 4°C), the supernatants were collected. The remaining cell pellets were resuspended in sample solvent (4.6% SDS, 10% β-mercaptoethanol, 0.124 M Tris, and 20% glycerol; pH 6.9), sonicated four times for 15 s each (Branson Sonifier), and centrifuged (16100 × g for 20 min at 4°C)
Saracatinib in vitro to collect the supernatant (representing intracellular protein fractions). Protein concentrations were adjusted using the bicinchoninic acid assay (BCA; find more Pierce) and separated by SDS-PAGE (10% or 12% acrylamide; Bio-Rad, Hercules, CA). The proteins were blotted onto Immobilon-P membranes (Millipore, Bedford, MA) and blocked with 5% skimmed milk for 1 h at room temperature Stattic manufacturer (RT). The membranes were washed with PBST (PBS containing 0.05% Triton X-100), immunoprobed sequentially with the MAbs, and incubated with HRP-conjugated goat anti-mouse polyvalent antibody (Sigma). Antibody-reactive bands were visualized following treatment with a chemiluminescence substrate system (ECL kit; Thermo Fisher Scientific, Rockford, IL) or DAB (6 mg of 3.3′-diaminobenzidine tetrahydrochloride; 10 μL of H2O2, 30%; 9 mL of 50 mM Tris–HCl, pH 7.6; 1 mL of 0.3% NiCl2). Two MAb-producing clones were selected for further study: L. monocytogenes (InlA-reactive)-specific
MAb-2D12 and Listeria genus-specific (p30-reactive) MAb-3F8. Immunofluorescence microscopy L. monocytogenes (serotypes 4b, 1/2a, 1/2b, and 4d) and L. innocua cell pellets (grown in 10 mL of LEB) were washed twice with
PBS and resuspended in 1 mL of PBS containing 5% bovine serum albumin (PBS-BSA). Subsequently, 20 μL of cells were incubated with MAbs diluted in 500 μL PBS-BSA for 1 h at 37°C. After washing with PBS (2×), the Mannose-binding protein-associated serine protease cell pellets were resuspended in 250 μL of FITC-conjugated goat anti-mouse IgG (1:100; Sigma) and incubated at 37°C for 1 h. After three sequential washes with PBS, the pellets were stained with Hoechst 33258 (for nuclear staining) for 15 min, and a single drop of the suspension was examined using an epifluorescence microscope (Leica, Buffalo Grove, IL). Antibody labeling For use with a fiber-optic sensor and magnetic beads that are pre-coated with streptavidin, affinity-purified antibodies were biotinylated using the EZ-Link Sulfo NHS-Biotinylation Kit (Pierce) as per the manufacturer’s instructions. The biotinylated MAbs were tested by ELISA in avidin-coated microtiter plates, and the ratio of biotin incorporated into the MAbs was calculated using the HABA assay (4′-hydroxyazoben-zene-2-carboxylic acid; Pierce). For use with a fiber-optic sensor, MAbs were also labeled with Cy5 using the Cy5-Ab labeling kit (Amersham Biosciences) as per the manufacturer’s protocol.