Nonlinear ipg strips

Protein extracts were quantified using bovine serum albumin (BioRad) as a standard. Mycelial and extracellular protein samples (80 and 50 µg, respectively) were loaded in precast 13 cm non-linear IPG strips (GE Healthcare) (pH 3–10 NL and 3–5.6 NL, respectively). Protein isoelectric focusing, SDS-PAGE, staining, image acquisition, and analysis were done as previously reported (Martins et al., 2013 (link), 2014b (link)). The gels were stained with flamingo dye (BioRad, USA) for image acquisition but for spot excision colloidal coomassie blue (Fluka, Switzerland) was used instead and higher quantities of protein were loaded, namely, 180 and 100 µg for mycelial and extracellular protein fractions, respectively. For each condition, three biological replicates (each accounting three batch cultures) were done.

For each gel, Cy2-, Cy3-, and Cy5-labeled proteins (50 µg each) were combined and an equal volume of rehydration buffer (8 M urea, 4% w/v CHAPS, 130 mM DTT, and 2% v/v Pharmalyte™ pH 3–10) was added. The pooled protein samples were subjected to isoelectric focusing carried out on nonlinear IPG strips, length 13 cm, pH 3–10 (GE Healthcare), rehydrated at 30 V for 12 h at room temperature. Isoelectric focusing was conducted at 500 V for 1 h, followed by 1000 V for 1 h, then 8000 V for 3 h and held at 8000 V to reach a total of 40000 Vh at 20°C and a maximum current setting of 50 µA per strip using Ettan IPG-phor apparatus (GE Healthcare). After IEF, individual strips were incubated in equilibration buffer (50 mM Tris-HCl, 6 M urea, 30% glycerol, 2% SDS) supplemented with 1% DTT. This step was repeated using the same buffer with 4% iodoacetamide in place of 1% DTT. The proteins were then resolved in 12.5% SDS-PAGE gels using the Hoefer SE 600 Ruby apparatus (GE Healthcare) at 15 mA for 15 min and then at 30 mA at 20°C, until the bromophenol blue dye front had run off the bottom of the gel. To facilitate MS analysis, 500 µg of the unlabeled pooled protein sample for each group was run in parallel on a preparative gel and was stained using Deep Purple staining (GE Healthcare) according to the manufacturer's instructions.

Individual 2-DE gel of single sample from one subject was run. The first dimension was performed on Ettan IPGphor II IEF system (GE Healthcare). 350 µl rehydration buffer (8Murea, 2%CHAPS, 0.5% IPG buffer (pH 3–10 NL), 1% Destreak reagent (GE Healthcare), and 0.002% bromphenol blue) containing 120 g protein were loaded onto nonlinear IPG strips (18 cm, pH 3–10 NL, GE Healthcare). The isoelectric focusing was performed at 50 V for 12 h linearly; 200 V for 1 h linearly; 500 V for 1 h linearly, 1000 V for 1 h linearly; 8000 V for 1 h rapidly; and finally achieved 60,000 Vh at the voltage of 8000 V. The strips were then equilibrated for 15 min in equilibration buffer containing 6M urea, 0.05M Tris-Cl (pH 8.8), 2% sodium dodecyl sulfate (SDS), 30% glycerol, and 1% DTT, and re-equilibrated for 15 min in the same buffer containing 2.5% iodoacetamide in place of DTT. The equilibrated gel strip was placed on the top of a 12.5% SDS-polyacrylamide gel electrophoresis (PAGE) gel, and embed with 0.5% low-melt agarose (Sigma). Then the second dimension separation was performed in an Ettan DALTsix Electrophoresis System (GE Healthcare) at 16°C as follows: 2.5 W/gel constant powers for 45 min, and then 17 W/gel constant powers until the bromophenol blue front reached the bottom of the gels.