Gs710 densitometer

2D-Polyacrylamide gel electrophoresis (PAGE) was performed as described previously [27 (link), 28 (link)]. Aliquots in sample buffer (7 M urea, 2M thiourea, 4.5% CHAPS, 100 mM DTE, 40 mM Tris, pH 8.8) were applied to the immobilized non-linear gradient (pH 3–10) strips (Amersham Biosciences, Uppsala, Sweden). Isoelectric focusing was performed at 80,000 Vh (volt-hour). The second dimension was analyzed on 9%–16% linear gradient polyacrylamide gels (18 cm × 20 cm × 1.5 mm) at a constant 40 mA per gel for approximately 5 h. After protein fixation in 40% methanol and 5% phosphoric acid for 1 h, the gels were stained with Coomassie Brilliant Blue G-250 for 12 h. The gels were then destained with H₂O, scanned in a Bio-Rad (Richmond, CA) GS710 densitometer, and converted into electronic files, which were then analyzed with Image Master Platinum 5.0 (Amersham Biosciences).

2-DE was carried out as described. Aliquots in sample buffer (7 M Urea, 2 M thiourea, 4.5% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 100 mM DTT, 40 mM Tris, pH 8.8, and trace of bromophenol blue) were applied to an immobilized pH 3–10 nonlinear gradient strip (Amersham Biosciences, Uppsala, Sweden). The samples (1 mg) were loaded. IEF was performed at 80,000 Vh. Then, 9%–16% linear gradient polyacrylamide gels electrophoresis (18 × 20 cm, 1.5 mm) was performed at a constant 40 mA, for approximately 5 h. The gels were fixed in 40% methanol and 5% phosphoric acid for 1 h and stained with Coomassie Brilliant Blue (CBB) G-250 for 12 h. The gels were destained with water, scanned in a Bio-Rad (Richmond, CA, USA) GS710 densitometer, and then analyzed using the Image Mater Platinum 5.0 image analysis program (Amersham Biosciences).