Nonlinear gradient strips

All 2-DE was performed as described [14 (link)]. Briefly, aliquots in sample buffer (260 μg) were applied to immobilized pH 3–10 nonlinear gradient strips (Amersham Biosciences, Uppsala, Sweden). Isoelectric focusing was performed at 80,000 Vh. The second dimension was analyzed on 9–16% linear gradient polyacrylamide gels (18 cm × 20 cm × 1.5 cm) at a constant 40 mA per gel for 5 hours. After protein fixation in 40% methanol and 5% phosphoric acid for 1 hour, gels were stained with Coomassie Brilliant Blue G-250 for 12 hours. Gels were then destained with H₂O, scanned in a Bio-Rad GS710 densitometer (Bio-Rad, Hercules, CA, USA), and converted into electronic files (12 bit tiff).

As previously described [21 (link),22 (link)], 2-DE was performed. Briefly, from each group, 1 mg of hippocampal protein was rehydrated in the sample buffer (7 M urea, 2 M thiourea, 4.5% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, 100 mM dithioerythritol, 40 mM Tris, pH 8.8), and applied to immobilized pH 3–10 non-linear gradient strips (Amersham Biosciences, Uppsala, Sweden). Isoelectric focusing was performed at 80,000 Vh. The strips were reduced and alkylated in Tributylphosphine equilibration buffer (6 M urea, 2% sodium dodecyl sulfate (SDS), 30 mM Tris, 20% glycerol, 2.5% acrylamide solution, and 5 mM Tributylphosphine), and then proteins were separated in the second dimension using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (9–16%) at 40 mA for 5 h. Proteins were fixed in 40% methanol and 5% phosphoric acid for 1 h, and the gels were stained with Coomassie brilliant blue G 250 solution overnight. The gels were destained with ultrapure distilled water. After fixation, the gels were scanned using a GS710 scanning densitometer (Bio-Rad, Richmond, CA, USA), and the results were converted into electronic files. The data were analyzed with the Image Master Platinum 5.0 image analysis program (Amersham Biosciences).