Ready prep overlay agarose

The raw clam extract was suspended in rehydration buffer containing 8 M urea, 50 mM DTT, 4% chaps, 0.2% carrier ampholyte, pH 3–10, and 0.0002% bromophenol blue. 50 μg protein sample was then applied to an immobilized nonlinear pH 3–10 gradient strip of 7 cm length (Bio-Rad, USA) for rehydration overnight (12–14 hours). Isoelectric focusing was run using the Protean IEF Cell Apparatus (Bio-Rad) with the following voltage/time gradient: 100 V for 1 minute, 250 V for 30 minutes, 4 000 V for 2 hours, and 4 000 V for 10 000 V-hr (Vhour). Before transferring the immobilized pH gradient (IPG) strip onto the second dimension, the strip was equilibrated sequentially for 10 minutes in a buffer containing 65 mM dithiothreitol and then 135 mM iodoacetamide in 125 mM Tris-HCl, pH 6.8, 6 M urea, 2% SDS, 30% glycerol, and 0.01% bromophenol blue. After equilibration, the strips were placed onto 12% SDS-PAGE separating gels with 5% of stacking gels and sealed in place using Ready Prep Overlay Agarose (Bio-Rad) for second dimension. The resultant gels were either stained for protein with Coomassie Brilliant Blue R-250 (Bio-Rad), scanned using Imaging Densitometer GS800 and analysed using PDQuest software (Bio-Rad), or subjected to protein transfer and immunoblotting as described above for SDS-PAGE and immunoblotting.

Immobilized pH gradient (IPG) strips (ReadyStrip IPG 11 cm, pH 4–7; Bio-Rad) were rehydrated in 200 µl of rehydration/sample buffer (Bio-Rad) containing 200 µg of the C. gattii CW or CP protein preparation. Isoelectric focusing (IEF) was carried out using PROTEAN IEF (Bio-Rad) under the following conditions: Step 1, 250 V for 20 min.; Step 2, ramped to 8000 V over 2.5 h, and Step 3, 8000 for a total of 30,000 V/h. Strips were then placed into equilibration buffer (Bio-Rad; 6 M urea, 2% SDS, 375 mM Tris-HCl pH 8.8, 20% glycerol, 2% DTT) for 15 min. Disulfide groups were subsequently alkylated by 10-min treatment with equilibration buffer of the same composition but using 2.5% w/v iodacetamide instead of DTT. Equilibrated IPG strips were then drained and placed on the top of 12.5% SDS-PAGE Criterion Precast Gels (Bio-Rad) and fixed using hot ReadyPrep Overlay agarose (Bio-Rad). The separation of proteins in the second dimension was carried out for 55 min at 200 V in Tris/glycine/SDS (TGS) running buffer (Bio-Rad) using Criterion electrophoresis equipment (Bio-Rad). Proteins in the gels were stained using SYPRO Ruby (Molecular Probes, Inc.) or, alternatively, transferred to PVDF membranes for immunoblot analysis.