A voltage gradient was applied (total of 40 kVh within 10 h, 50 μA/IPG strip). Prior to SDS-PAGE, the IPG
strips were equilibrated in gel loading buffer for 10 min (120 mM Tris pH 6.8, 20% (v/v) glycerol, 4% (w/v) SDS, 200 mM DTT and traces of bromphenol blue). The second dimension-electrophoresis was carried CBL0137 out at 10°C using 12%-acrylamide gels (18 × 18 cm). Gel analysis Protein spots were visualized with a Typhoon™ 9400 Series Variable Mode Imager (Amersham Pharmacia Biotech). The resulting gel images were processed using DeCyder Differential Analysis Software v5.02 (Amersham Pharmacia Biotech). Protein spots were detected using the Differential In-gel Analysis (DIA) mode of ‘DeCyder’. The Biological Variation Analysis (BVA) mode allowed inter-gel matching on the basis of the in-gel standards (Cy2). Spot selleck products intensities were normalized to the internal standard. For each spot, averages and click here standard deviations of protein abundance were compared between the profiles of B. suis grown in rich medium and cultivated under starvation conditions. The Student’s t-test was applied to each set of matched spots. Significantly regulated proteins (p-value ≤ 0.05) were then identified by mass spectral analysis. To exclude
non-real spots prior to MALDI-TOF analysis, the three-dimensional displays of significant spots were also checked manually. Protein identification by mass spectral analysis Prior to spot-picking, 2D gels were stained with Coomassie to ensure that the majority of the unlabeled molecules of the proteins of interest were recovered for MALDI-MS analysis. Protein spots of interest
were manually picked and washed three times in 50 mM (NH4)2HCO3. Then, gel spots were dehydrated in 100% acetonitril for 5 min. After removal of the Nabilone supernatant, 1 μl protease-solution (0.05 μg/μl trypsin in 10 mM (NH4)2HCO3) was added and allowed to penetrate into the gel. Another 5–10 μl NH4HCO3-buffer (10 mM, in 30% acetonitril) were added to the gel plugs which were incubated overnight at 37°C for digestion. The samples were desalted in C18-ZipTips™ (Millipore, Bedford, MA, USA) according to manufacturer’s instructions. The desalted and concentrated peptides were eluted from the ZipTips™ on the MALDI targets with matrix solution (0.1% trifluoroacetic acid (TFA)/80% acetonitrile, equally mixed with 2,5-dihydroxybenzoic acid: 2-hydroxy-5-methoxybenzoic acid, 9:1). For analysis of the tryptic peptides, MALDI-TOF mass spectrometry was carried out using the Voyager-DE™ STR Biospectrometry Workstation (Applied Biosystems). The spectra were acquired in a positive reflectron mode (20 kV) and collected within the mass range of 700 to 4,200 Da. The autolytic fragments of trypsin acted as internal calibrants. The peptide mass fingerprint spectra were processed with the Data Explorer v4.9 Software (AB Sciex).