Two Dimensional Gel Electrophoresis

3.3.1. Isoelectric Focusing

Three-hundred micrograms of each tissue sample were applied for the first-dimensional isoelectric focusing (IEF) on 7-cm, immobilized, pH 3-10 linear gradient strips (Bio-Rad, Hercules, CA) at 20°C and a 50 ^A/strip. Isoelectric focusing (IEF) was run in three steps: 500 V for 1 hr, 1000 V for 1 hr, and 8000 V for 3 hr.

3.3.2. Sodium Dodecyl Sulfate-Polyacrilamide Gel Electrophoresis (SDS-PAGE)

The second-dimensional run was performed on precast polyacrylamide gels (2-D homogeneous 12.5; GE Healthcare) in two steps: (i) 600 V, 20mA for 30min, and (ii) 600 V, 50 mA for 70min. After electrophoresis, the blotting gels were used for 2-D immunoblotting, and the staining gels were stained with CBB R250 (Nacalai Tesque, Kyoto, Japan) for 24 hr. Gels were destained with 10% acetic acid in water containing 30% methanol for 30min and then with 7% acetic acid and used for in-gel digestion.

3.3.3. Two-Dimensional Immunoblot Analysis

For the blotting-gels, fractionated proteins were transferred electrophoretically onto PVDF membranes (Immobilon-P, Millipore Corporation, Bedford, MA), and the membranes were blocked for 1 hr at 4°C with TBS containing 1% skim milk. They were subsequently incubated overnight at 4°C with autologous serum (1:50 dilution), washed four times with TBS containing 0.05% Tween 20, and incubated for 1hr at 4°C with horseradish peroxidase-conjugated secondary antibody (1:1000, #55256; ICN Pharmaceuticals, Aurora, OH). The reaction was visualized with a chemiluminescence reagent (ECL; Amersham Biosciences, Buckinghamshire, UK).

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