Pdquest basic 8.0 program

Isoelectric focusing (IEF): IEF was performed as described previously [34] (link). Briefly, each pH 3–10 IPG strip (17 cm, NL) or pH 4–7 IPG strip (7 cm, NL) was rehydrated at RT for 12 h with 350 µg or 100 µg M. bovis protein sample in 400 µl or 150 µl rehydration sample buffer (6 M urea, 2 M thiourea, 4% (w/v) CHAPS, 65 mM DTT, 0.5% (v/v) IPG, 0.04% (w/v) Bromophenol blue and 40 mM Tris-base, pH 9.6). IEF was performed in a PROTEAN® IEF System (Bio-Rad, U.S.). The parameters used for IEF were as follows: 50 V for 2 h, 500 V for 30 min, 1000 V for 30 min, 8000 V for 4 h. The final phase of 8000 V was terminated after 50,000 Vh.
SDS-PAGE: After IEF, the IPG strips were successively equilibrated for 15 min in equilibration buffer I containing 64.8 mM DTT and buffer II containing 135 mM iodoacetamide. Two equilibrated IPG strips were subjected to 12% polyacrylamide gel electrophoresis and sealed with 0.5% agarose solution. The second dimension was carried out at 50 V for 3 h followed by 100 V for 15 h. One strip gel was stained with Coomassie brilliant blue R-350, and the other was subjected to immunoblotting. The stained gel was scanned with an Image Scanner (Amersham Biosciences, U.S.) and analyzed using the PDQuest Basic 8.0 program (Bio-Rad). Three replicates were performed.

For 2-DE, 380 μg and 750 μg of proteins were loaded onto analytical and preparative gels, respectively [20 , 24 (link)]. The IPG strips were rehydrated for 16 h in 400 μl of rehydration buffer containing the protein samples. IEF was performed in five steps: 150V for 3 h, 300 V for 3 h, 1000V (gradient) for 6 h, 10000V (gradient) for 3 h, and 10000V for 60 kVh. The gel strips were equilibrated with 1% DTT and 4% iodoacetamide in equilibration buffer. The strips were then subjected to the second-dimensional electrophoresis onto 10% SDS-polyacrylamide gels. Three replicates were performed for each sample. Protein spots in the analytical gels and preparative gels were stained with coomassie brilliant blue R-250 (CBB R-250- Bio-Rad, USA) and scanned by GS-800 Calibrated Densitometer (Bio-Rad) and image analysis was accomplished using PD Quest Basic 8.0 program (Bio-Rad, USA). Each paired spot was manually verified to ensure a high level of reproducibility between normalized spot volumes of gels produced in triplicate data. The proteins of the other gels were subjected to immunoblotting assay.