2.10 Western blot analysis
SDS-PAGE using a 4-20% gradient
gel (Mini-PROTEAN precast gels, Bio-Rad, Hercules, CA) under reducing
conditions with 5% β-mercaptoethanol was performed at 200 V for 37
mins. Then the gel was transferred to a 0.45 µm nitrocellulose membrane
for 90 mins at 100 V and blocked with 5% non-fat dry milk (NFDM) in
PBST overnight. The blotted nitrocellulose membrane was washed with PBST
buffer three times at 5-min intervals and incubated with the mouse BChE
(D5) antibody HRP (Santa Cruz Biotechnology, Dallas, TX) diluted in 10
mL 5% NFDM at a 1:1,000 dilution for 1 h at room temperature on a
rotary shaker at 60 rpm. The blot was then washed with PBST buffer three
times for 5 min each. Clarity enhanced chemiluminescence (ECL) substrate
(1 mL) was dropped over the blot to detect BChE bands using
chemiluminescent mode in the ChemiDoc system (Bio-Rad, Hercules, CA).
Human BChE derived from blood plasma, kindly provided by Dr. Douglas M.
Cerasoli (US Army Medical Research Institute of Chemical Defense,
USAMRICD), was used as a control.