A 4.0–7.0 pH
gradient was used. Gel prefocusing was carried out according to the
following program: 200V for 15 min, 300V for 15 min and 400V for
15 min. Isoelectric focusing (IEF) was performed at 400V for 20 h,
to complete 8000Vh. After IEF, the gels were extruded from the
glass capillary tubes and equilibrated immediately in 2 mL of equilibration
solution (10% glycerol, 5% -mercaptoethanol, 2.3% SDS,
0.0625 M Tris–HCl pH 6.8) for 10 min. V