Ining 5 mM EDTA and 20 mg lysozyme, the sample was incubated for

Ining 5 mM EDTA and 20 mg lysozyme, the sample was incubated for 30 min at room temperature, spheroplasts were collected by centrifugation at 10.000 g for 20 min and the supernatant was used as the periplasmic fraction. Spheroplasts were disrupted by sonication (Sonifier W250; Branson) in 240 ml 100 mM Tris-HCl (pH 8). After centrifugation for 5 min at 5,0006g to remove undisrupted cells and cell debris, the total membrane fraction was collected by centrifugation for 45 min at 13,0006g and the supernatant was used as the cytoplasmic fraction. An amount equivalent to a cell density of an O.D.580 nm of 0.5 of each fraction was used for Western blotting.20 mM mannose in 100 mM Tris-HCL (pH 8.0). As a negative control, the same experiment was carried out with the lecBdeficient P. aeruginosa mutant PATI2. One ml each of the eluates was analyzed by SDS-PAGE, 2-D-gel electrophoresis and MALDI-TOF mass spectrometry.Isolation of LecB Ligands from the Outer MembraneThe isolation procedure was carried out at 37uC. The outer membrane fraction was incubated in 100 mM Tris-HCl containing 2 mg His-tagged LecB for 1 h. After loading the sample onto a Ni-NTA-agarose column (Quiagen, volume 5 ml)), the column was washed with 50 ml Tris-HCl (pH 8.0) containing 50 mM imidazole and 300 mM NaCl to remove non-specifically bound proteins. LecB binding proteins were eluted with 5 ml 100 mM Tris-HCl containing 20 mM L-fucose. 1 ml of the sample was analyzed by 2-D-gel electrophoresis and MALDI-TOF 25837696 mass spectrometry.Outer Membrane IsolationOuter membranes were isolated by a modification of the method described previously [41]. P. aeruginosa PATI2 cells (500 mg dry weight) were harvested after growth for 48 h at 37uC by centrifugation at 3000 g for 10 min. The cells were resuspended in 200 ml 100 mM Tris-HCl (pH 8) containing 10 mg lysozyme, incubated for 30 min at 37uC and disrupted by three passages 78919-13-8 KS 176 site chemical information through a French press. Intact cells were separated from the cell extract by centrifugation at 5,0006g for 10 min. The supernatant was centrifuged at 13,0006g for 1 h. The pellet, consisting of the total membrane fraction, was resuspended in 10 ml 100 mM Tris-HCl (pH 8) containing 2 lauryl sarcosinate and incubated at room temperature for 20 min. After centrifugation for 40 min to at 45.000 g the pellet consisting of the outer membrane fraction was resuspended in 100 mM Tris-HCl (pH 8.0).SDS-PAGE and 2 D Gel ElectrophoresisPrior to SDS-PAGE, samples were suspended in SDS-PAGE sample buffer, boiled for 5 min at 99uC and loaded onto an SDS16 polyacrylamide gel. SDS-gel electrophoresis was run for 1 h at 200 V. For 2 D gel electrophoresis, the proteins were precipitated overnight with 20 (v/v) TCA and afterwards washed twice with acetone. The protein preparation was air dried and resuspended in 1 ml rehydration buffer (7 M urea, 2 M thiourea, 4 (w/v) 3-[(3-cholamidopropyl) dimethylammonio]-1propanesulfonate (CHAPS), 2 IPG buffer and pH 3?1 negative-logarithmic stripes as recommended by the manufacturer (GE-Healthcare, Freiburg, Germany), 1 (v/v) bromphenol blue). Protein was loaded onto an IPG strip and isoelectric focusing was performed at a maximum voltage of 8,000 V. The second dimension SDS-gel electrophoresis was run for 3 h in a 12.5 polyacrylamide gel at 250 V. Afterwards, the gels were stained with Coomassie Brilliant Blue G250.Isolation of LecB Ligands by Affinity Chromatography on D-mannose-agaroseP. aeruginosa PAO1 was grown in 0.5 l NB-medium at 37uC.Ining 5 mM EDTA and 20 mg lysozyme, the sample was incubated for 30 min at room temperature, spheroplasts were collected by centrifugation at 10.000 g for 20 min and the supernatant was used as the periplasmic fraction. Spheroplasts were disrupted by sonication (Sonifier W250; Branson) in 240 ml 100 mM Tris-HCl (pH 8). After centrifugation for 5 min at 5,0006g to remove undisrupted cells and cell debris, the total membrane fraction was collected by centrifugation for 45 min at 13,0006g and the supernatant was used as the cytoplasmic fraction. An amount equivalent to a cell density of an O.D.580 nm of 0.5 of each fraction was used for Western blotting.20 mM mannose in 100 mM Tris-HCL (pH 8.0). As a negative control, the same experiment was carried out with the lecBdeficient P. aeruginosa mutant PATI2. One ml each of the eluates was analyzed by SDS-PAGE, 2-D-gel electrophoresis and MALDI-TOF mass spectrometry.Isolation of LecB Ligands from the Outer MembraneThe isolation procedure was carried out at 37uC. The outer membrane fraction was incubated in 100 mM Tris-HCl containing 2 mg His-tagged LecB for 1 h. After loading the sample onto a Ni-NTA-agarose column (Quiagen, volume 5 ml)), the column was washed with 50 ml Tris-HCl (pH 8.0) containing 50 mM imidazole and 300 mM NaCl to remove non-specifically bound proteins. LecB binding proteins were eluted with 5 ml 100 mM Tris-HCl containing 20 mM L-fucose. 1 ml of the sample was analyzed by 2-D-gel electrophoresis and MALDI-TOF 25837696 mass spectrometry.Outer Membrane IsolationOuter membranes were isolated by a modification of the method described previously [41]. P. aeruginosa PATI2 cells (500 mg dry weight) were harvested after growth for 48 h at 37uC by centrifugation at 3000 g for 10 min. The cells were resuspended in 200 ml 100 mM Tris-HCl (pH 8) containing 10 mg lysozyme, incubated for 30 min at 37uC and disrupted by three passages through a French press. Intact cells were separated from the cell extract by centrifugation at 5,0006g for 10 min. The supernatant was centrifuged at 13,0006g for 1 h. The pellet, consisting of the total membrane fraction, was resuspended in 10 ml 100 mM Tris-HCl (pH 8) containing 2 lauryl sarcosinate and incubated at room temperature for 20 min. After centrifugation for 40 min to at 45.000 g the pellet consisting of the outer membrane fraction was resuspended in 100 mM Tris-HCl (pH 8.0).SDS-PAGE and 2 D Gel ElectrophoresisPrior to SDS-PAGE, samples were suspended in SDS-PAGE sample buffer, boiled for 5 min at 99uC and loaded onto an SDS16 polyacrylamide gel. SDS-gel electrophoresis was run for 1 h at 200 V. For 2 D gel electrophoresis, the proteins were precipitated overnight with 20 (v/v) TCA and afterwards washed twice with acetone. The protein preparation was air dried and resuspended in 1 ml rehydration buffer (7 M urea, 2 M thiourea, 4 (w/v) 3-[(3-cholamidopropyl) dimethylammonio]-1propanesulfonate (CHAPS), 2 IPG buffer and pH 3?1 negative-logarithmic stripes as recommended by the manufacturer (GE-Healthcare, Freiburg, Germany), 1 (v/v) bromphenol blue). Protein was loaded onto an IPG strip and isoelectric focusing was performed at a maximum voltage of 8,000 V. The second dimension SDS-gel electrophoresis was run for 3 h in a 12.5 polyacrylamide gel at 250 V. Afterwards, the gels were stained with Coomassie Brilliant Blue G250.Isolation of LecB Ligands by Affinity Chromatography on D-mannose-agaroseP. aeruginosa PAO1 was grown in 0.5 l NB-medium at 37uC.