Equilibration buffer

Total trichomonad protein extracts were obtained from 2 × 10⁷ parasites by 10% trichloroacetic acid (TCA) precipitation at 4 °C overnight [47 (link)]. The protein pellet was resuspended in sample buffer, boiled for 5 min, and analyzed by 1D SDS-PAGE (1DE) on 12% polyacrylamide gels. For 2DE, we used TCA-precipitated proteins from 1 × 10⁷ parasites. The protein pellet was washed with PBS and cold acetone, air-dried, resuspended in 150 µL Rehydration Buffer (Bio-Rad Laboratories, Hercules, CA, USA), centrifuged, and the supernatant was loaded into 7 cm ReadyStrip IPG strips (linear pH gradient 4–7; Bio-Rad) and actively rehydrated for 16 h at 20 °C and 50 V. Protein isoelectric focusing (IEF) was performed using a protean IEF cell (Bio-Rad) in four steps: 250 V for 20 min, 4000 V for 3 h, a gradual increase from 4000 V to 10,000 V-h and holding at 500 V. After IEF, the IPG strips were equilibrated for reduction and alkylation in equilibration buffer (Bio-Rad). For the second dimension, the proteins were resolved by SDS-PAGE on 12% polyacrylamide gels. The protein gels were Coomassie Brilliant Blue (CBB) stained or transferred onto NC membranes (Bio-Rad) for WB detection.

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.