The ammonium shock was achieved Gefitinib solubility dmso by adding ammonium chloride to 1 mM of the solution. Cellular fractions were obtained as follows: 50 mL of the culture was withdrawn before or 5 min after the ammonium shock. The culture was cooled by immersion in liquid nitrogen and the cells were harvested by centrifugation (5000 g for 5 min at 4 °C). The cells were resuspended in 1 mL of SP buffer (40 mM K2HPO4, 22 mM KH2PO4, 150 mM NaCl, pH 7.2) and processed exactly as described (Huergo et al. 2006). Membrane preparations were suspended in 6 M urea, 2 M thiourea, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate 4% (w/v), Pharmalyte pH 4–7, 0.5% (v/v) and 20 mM dithiothreitol.
For isoelectrofocusing, 500 μg of protein was loaded onto a 13 cm, pH 4–7 Selleckchem Torin 1 linear IPG strip (GE Healthcare). After rehydration, the isoelectrofocusing run was performed until the accumulation of 56 kVh, following the manufacturer’s instructions (GE Healthcare). The second dimension was achieved by 11% sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE). Gels were stained with colloidal Coomassie blue and images were analyzed using imagemaster platinum 6.0. The signal of each spot was normalized using the total density of the gel image. The experiments reported here were reproduced in two different biological replicates and two technical 2D-PAGE repetitions from the same sample. The spots were excised from the gels (ammonium shock treatment) and destained for 1 h in a solution of acetonitrile
50% (v/v) and 20 mM ammonium bicarbonate. Resveratrol The gel piece was immersed in pure acetonitrile for 5 min, the acetonitrile removed and the gel dried under air. In-gel digestion was performed using 0.1 μg of sequencing-grade trypsin in acetonitrile 10% (v/v) and 20 mM ammonium bicarbonate. After overnight incubation at 37 °C, aliquots of each digested sample were mixed with a saturated matrix solution of α-cyano-4-hydroxycinnamic acid (in acetonitrile 50% v/v, TFA 0.1% v/v), spotted onto the MALDI target and allowed to dry. Mass spectra were acquired using a MALDI-TOF/TOF Autoflex II (Bruker Daltonics). MS analyses were performed in a positive ion reflection mode using an accelerating voltage of 20 kV. MS/MS analyses were performed in a positive ion LIFT reflection mode. Peak lists were created using flexanalysis 3.0 software (Bruker Daltonics). Trypsin autolysis signals (842.5 and 2211.1) were used as internal standards when present. A database search was performed using mascot 2.2. Mass lists were searched against a database of H. seropedicae predicted proteins. Carbamidomethylation of cysteines was set as fixed and oxidation of methionine as variable modifications. Error tolerance was 100 p.p.m. for peptide mass fingerprint (PMF) search and for MS/MS parent ion. MS/MS ion search error was set as 0.3 Da. signalp 3.0 (Bendtsen et al., 2004) was used for prediction of Sec signal peptides. tatp 1.0 (Bendtsen et al., 2005b) was used to predict TAT-dependent signal peptides.