2D-page

24 cm pH 4.9–5.7 narrow range IEF strips (BioRad) were passively rehydrated with a mixture of the labelled sample and 472μL of rehydration buffer (8 M urea, 4% CHAPS, 1% Destreak™ (GE Healthcare), 1% pH 3–11 IPG buffer (GE Healthcare), and a trace of bromophenol blue (Sigma-Aldrich) under biotechnology grade mineral oil (Bio-Rad), for nine hours at 4°C, prior to isoelectric focusing. Isoelectric focusing was performed on an Ettan™ IPGphor II™ ceramic manifold, according to the follow protocol: Step 500 V for 1000 VHrs; linear gradient to 1000 V over 1500 VHrs hours; linear gradient to 10,000 V over 16,500 VHrs hours; step at 10,000 V for 90,000 VHrs and held at 50 V to prevent defocusing. After focusing, the strips were gently rocked in the dark for 15 min in 10 ml of 1% DTT (Bio-Rad) in equilibration buffer (6 M urea, 2% SDS, 20% glycerol, 50 mM Tris-HCl pH 8.8), followed by 15 min in 10 ml of 2.5% iodoacetamide (Sigma-Aldrich) in equilibration buffer. For the second dimension PAGE, the focused strips were sealed in agarose with a trace of bromophenol blue onto 24 cm ×1.5 mm, 11% acrylamide gels. PAGE was run on a water-cooled DALT6 tank for 1 h at 2 W per gel, followed by approximately 5 h at 15 W per gel, until the blue dye front migrated to the bottom of the gels. Gels were scanned, at 100μm resolution, for fluorescence using a Typhoon™ Trio scanner (GE Healthcare). Photomultiplier tube voltages were selected to assure sub-saturating intensities of protein spots. Scans of the blue emitting JAV-I-187 labeled proteins used the 488 nm excitation laser and an HQ 510 nm, 30 nm bandpass emission filter (Chroma Technology). For the green emitting BDR-I-227 labeled proteins, the 532 nm excitation laser was used with a 580 nm, 30 nm bandpass emission filter (GE Healthcare).