Mini protean cell

Salivary gland protein samples were immobilized on IPG strips (17 cm) of pH 3–10 (Linear, Bio-Rad) using a Protean IEF Cell (Bio-Rad) and kept for isoelectric focusing with a default cell temperature of 20°C and a maximum current of 50 mA/strip. Briefly, 300 μl sample in rehydration buffer were thawed and each of them were pipetted as a line along the edge of a channel. IPG strips were gently placed (gel side up) on to the sample and finally layered with 2–3 ml of mineral oil. The sample supernatants were rehydrated on IPG strips at 20°C overnight after which IEF was run in three steps: 1. 250V for 20 min Linear; 2. 10000V for 2.5 hrs Linear; 3. 10000V for 5–7 hrs and 40000 V-hrs in Rapid mode. After IEF run the strips were equilibrated in equilibration buffer I (6M urea, 0.375M Tris-HCl, pH 8.8, 2% SDS, 20% glycerol and 2% DTT) and equilibration buffer II (6M urea, 0.375M Tris-HCl, pH 8.8, 2% SDS, 20% glycerol and 2.5% iodoacetamide for 10 min. The second dimension was carried out on 12% SDS-PAGE on Mini Protean cell (Bio-Rad). The 2D gels were silver stained with FOCUS-FAST silver^TM stain (G-Biosciences) after the run to visualize the spots. Stained gels of both the susceptible and resistant strains were scanned and analyzed using ImageMaster 2D Platinum 7.0 software (GE Healthcare Life Sciences).

The ES proteins were precipitated using trichloroacetic acid (TCA) and acetone using a previously described method with some modifications [27 (link)]. The electrophoresis was performed as described previously [17 (link),28 (link)]. Briefly, 300 or 200 μg of ES proteins were loaded onto 11-cm pH 4–7 immobilized pH gradient (IPG) strips (Bio-Rad, USA) and separated by isoelectric focusing (IEF). IEF was performed using a Protean IEF Cell at 20°C as follows: S1: 50 V, 12 h; S2:250 V, 30 min; S3: 1 000 V, 30 min; S4: 8 000 V, 4 h; and S5: 8 000 V, 40 000 Vh (using a limit of 50 μA/strip). SDS-PAGE was performed with 10% gels using a Mini Protean cell (Bio-Rad, USA). Three replicates were run for the sample. After 2D gel electrophoresis, proteins were either stained with Coomassie blue R-250 for proteomic analysis or used for immunoblotting as previously described [29 (link)]. Both the 2-DE and immunoblotting tests were repeated three times, with no variation in results observed. Images of immunoblots were captured using ImageScanner (GE healthcare, USA) and aligned with equivalent protein stained 2-DE gels using Image Master 2D Platinum 6.0 (GE healthcare, USA).

Proteins in plant extracts were analysed by SDS-PAGE electrophoresis in 12% gel using a Mini-Protean cell (Bio-Rad, USA) and transferred to 0.45-μm nitrocellulose membrane. Blots were incubated for 2 h in a blocking buffer and then overnight with polyclonal rabbit antibody raised to tomato GSNOR in 1:1000 dilution^{28 (link)}, or anti-APX polyclonal rabbit antibody in 1:2000 dilution (Agrisera, Sweden). The membranes were washed six times for 10 min in 0.1% Tween-20 in TBS and then incubated for 2 h with goat anti-rabbit IgG conjugated with horseradish peroxidase (Sigma-Aldrich, USA) in 1:10,000 dilution. The membranes were washed for 1 h in 0.1% Tween-20 in TBS and then incubated for 5 min with a Western blotting luminol reagent (Santa Cruz Biotechnology, USA). The chemiluminescence was detected with a photographic film (GE Healthcare, USA). Chemiluminescence signal intensities were assessed using ImageJ 1.33 software (National Institute of Health, USA).