18 cm immobiline dry strips

The vacuum-dried protein samples were dissolved in a lysis buffer containing 7 M urea, 65 mM dithiothreitol (DTT), 4% (w/v) CHAPS, 2 M thiourea, and 0.2% carrier ampholytes for 1 h at room temperature with vortexing every 10 min, the homogenate was centrifuged at 15,000 g for 20 min. The protein concentration of the supernatant was determined by Bradford assay with bovine serum albumin as the standard [42 (link)]. 2-DE was performed following the protocol described by Sheoran et al. [1 (link)] and Zhang et al. [26 (link)]. Briefly, Protein samples (600 µg) were diluted in a rehydration buffer for 12 h. Isoelectric focusing (IEF) was performed using the Ettan III system (GE Healthcare, Chicago, IL, USA) and 18 cm Immobiline Dry Strips (pH 4–7, GE Healthcare). After IEF, the strips were treated in an equilibration buffer, placed on top of the vertical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and sealed with agarose and bromophenol blue. The gels were run with a running buffer in a PROTEAN II XL multi-cell (Bio-Rad, Hercules, CA, USA) electrophoresis tank under 10 mA constant for 30 min, and then 30 mA until the tracking dye reached the bottom of the gels. Three representative gels per sample were used for further analysis.

Samples containing 600 µg of protein were resuspended in the rehydration buffer (7M urea; 2M thiourea; 2% w/v CHAPS; 10 mM DTT; 1% v/v IPG buffer pH 4–7; 0.002% bromophenol blue) to reach a final volume of 340 µL. The protein samples were loaded on 18-cm Immobiline DryStrips, pH 4–7 (GE Healthcare, Uppsala, Sweden), and rehydrated for 10 h (passive rehydration). Isoelectric focusing was performed with an IPGphor isoelectric focusing unit (GE Healthcare), and SDS-PAGE was run using the PROTEAN II XL Cell (Bio-Rad, Hercules, CA, USA) as described previously by Likszo et al. [23 (link)]. After electrophoresis, gels were fixed in methanol:acetic-acid:water (40:10:50) for 1 h and stained using a Coomassie Brilliant Blue G250 (Sigma Aldrich, Saint Louis, MO, USA).