Criterion dodeca cell

The soluble protein fraction from the newly acquired, virulent and attenuated N. perurans cultures were analysed in triplicate using 2D electrophoresis. Triplicate immobilised pH gradient strips (IPG, pH 4–7, 11 cm) were passively rehydrated overnight with 120 µg of protein in a final volume of 200 µL of re-hydration buffer 1. The strips were focused in a PROTEAN i12 IEF system (Bio-Rad) at a current limit of 50 Amp/IPG strip using a step voltage gradient (250 V for 20 min, stepped up to 8000 V maximum for 1 h; 26,000 V-h total) at 20 °C. Prior to the second dimension IPG strips were equilibrated in a reducing buffer (6 M urea, 0.375 M Tris–HCl pH 8.8, 2% (w/v) SDS, 20% (v/v) glycerol, 2% (w/v) dithiothreitol ) for 20 min and subsequently placed in an alkylating buffer (6 M urea, 0.375 M Tris–HCL pH 8.8, 2% (w/v) SDS, 20% (v/v) glycerol, 2.5% iodoacetamide) for 20 min. The second dimension was performed using AnykD Criterion TGX Precast gel in the Criterion Dodeca cell (Bio-Rad, CA) where IPG strips were electrophoresed for 40 min at 200 V. Gels were stained using QC Colloidal Coomassie (Bio-Rad) overnight and destained by washing in deionized water in triplicate, followed by incubation in wash buffer (10% ethanol [v/v], 7.5% acetic acid [v/v]).

Samples of PBDs were electrophoresed, as described by Sheng et al. [15 (link)], with some modifications. The samples were diluted in Milli-Q water to a final concentration of 2 μg/μL protein, according to the declared protein content provided on the product packages, and mixed 1:1 with the Laemmli sample buffer (20 mM Tris, 2% SDS, 20% Glycerol, bromophenol blue, 20 mM dithioerythritol) under reducing conditions. The samples were heated to 90 °C for 2 min and centrifuged shortly, and 20 μL were loaded in wells of polyacrylamide gel Criterion Protein Gel (TGX Stain-Free, any kDa, Bio-Rad Laboratories, Hercules, CA, USA) and 5 μL of ProteinLadder (Bio-Rad Laboratories, Hercules, CA, USA) was applied as a marker. The gels were run at 200 V for 30–40 min on a Criterion Dodeca Cell (Bio-Rad Laboratories, Hercules, CA, USA) with a Laemmli running buffer (250 mM Tris-Base, 1.89 M Glycine, 10% SDS). The gels were fixed in 50% ethanol, 8% phosphoric acid for at least 2 h and then stained with Coomassie Blue (5% in Milli-Q, w/v) [15 (link)]. The gels were photographed with ChemiDoc XRS+ and processed with ImageLab software version 6.0 (Bio-Rad Laboratories, Hercules, CA, USA).