, 2004). ROS was measured VX-809 purchase essentially as described by Ackerley et al. (2006) excepting that incubation with H2DCF-DA was carried out for 30 min, and fluorescence of the dye was measured by a Hitachi F-3010 spectrofluorometer (excitation at 485 nm and emission at 530 nm). The specificity of H2DCF for different ROS species is limited (Setsukinai et al., 2003), and our assay could detect hydrogen peroxide (H2O2), hydroxyl radical (˙OH), and superoxide anion (). TSB-6 cells were grown in LB at 37 °C without chromate till OD600 nm of 0.25. Then, one-half of these cells were heat stressed by transferring to 65 °C. Both the control and heat-stressed cells were grown for another 24 h.
The cells were harvested and soluble extracts prepared as described previously. Ammonium sulfate was then added to the soluble extracts to 90% saturation. The mixture was centrifuged at 12 000 rpm for 30 min and the supernatant discarded. The pellet was dissolved in 20 mM sodium phosphate buffer and dialyzed against 20 mM sodium phosphate buffer, pH 7.0. For the first-dimension electrophoresis, IPG strips of 7 cm length
and nonlinear pH range 4–7 (Bio-Rad) were rehydrated with 150 μg protein in 125 μL of rehydration buffer (provided with the kit) for 16 h. Isoelectric focusing was carried out in a PROTEAN IEF Cell (Bio-Rad) at 4 kV for 1 h with linear voltage amplification and finally to 20 kVh with rapid click here amplification. Before SDS-PAGE in the second dimension, of the focused strips were equilibrated
at room temperature first with a buffer containing 20% v/v glycerol, 0.375 M Tris–HCl, pH 8.8, 6 M urea, 2% (w/v) SDS, 130 mM DTT and then with a second buffer containing 20% (v/v) glycerol, 0.375 M Tris–HCl, pH 8.8, 6 M urea, 2% (w/v) SDS, and 135 mM iodoacetamide. Electrophoresis was carried out using 10% SDS polyacrylamide gels in a Mini-PROTEAN 3 system (Bio-Rad) at constant 200 V for 35 min. The gels were stained in 0.1% (w/v) Coomassie Brilliant Blue R-250. 2D gel images were obtained by VersaDoc™ (Model 4000) Imaging System (Bio-Rad). The spots were detected, analyzed, and assessed for reproducibility with PDQuest Advanced 2D Analysis software (version 8.0.1; Bio-Rad). Three independent experiments were performed with control and heat-stressed samples, and spots present in each of the three replicate gels of both samples were considered. Spots obtained from the control were taken as standard to determine the fold changes in the corresponding spots obtained from heat-stressed samples. Protein spots were excised from gels and subjected to in-gel digestion essentially as described by Shevchenko et al. (2006) using 25 ng μL−1 of trypsin and without the active extraction step. Mass spectrometry of the digested sample was carried out following a published protocol (Sinha & Chattopadhyay, 2011). Similarity searches to identify the proteins were performed using mascot search engine (version 3.5; Matrix Science, London, UK; www.matrixscience.com).