An 11.25% resolving gel was prepared according to Laemmli
(1970) containing 1.5 M Tris–HCl (pH 8.8), 20% SDS, 10% ammonium
persulphate and TEMED. The stacking gel (4.8%) was made
using 0.5 M Tris–HCl (pH 6.8), 20% SDS, 10% ammonium persulphate
and TEMED. The electrophoresis was accomplished at 150 V
for 9 h using Hoefer SE600 Ruby Standard Vertical Electrophoresis
Unit (Amersham Biosciences) with running buffer consisted 25 mM
Tris base, 192 mM glycine and 3 mM SDS. The Nacalai Tesque prestained
protein marker was used. The gel was stained with 0.5%
Coomassie Brilliant Blue R-250 in 45% (v/v) methanol, 10% (v/v)
acetic acid for one hour and destained for 12 h in 20% (v/v) methanol
and 10% (v/v) acetic acid. The gel was washed with distilled water
until the background was clear prior to view. Finally the gel was
viewed and photographed with Alpha Innotech Alphaimager HP
MultiImage II.