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- Prepare gel for electrophoretic separation of the protein mix-
ture. First, preparation of separation gel (10%): Add 4.1 mL
H 2 O, acrylamide/bis (30%) 3.3 mL, Tris–HCl (1.5 M,
pH 8.8) 2.5 mL, 10% SDS 100, 10 μL TEMED, 10% APS
32 μL. Then, preparation of stacking gel (4%): Add 6.1 mL
H 2 O, acrylamide/bis (30%) 1.3 mL, Tris–HCl (1.5 M,
pH 8.8) 2.5 mL, 10% SDS 100 μL, 10 μL TEMED, 10% APS
100 μL. The separation and stacking gel polymerize quickly.
After that, pour separation gel into the gel chamber and leave
~2 cm below the bottom of the comb for stacking gel. Make
sure there is no bubble. Put isopropanol on top of the gel.
This will remove the bubbles and keep the gel wet. Within
30 min, gel should be completely polymerized and then
remove the isopropanol and wash with distilled water. Pour
the stacking gel on top of separation gel. Place the comb to
make wells. In 30 min, the stacking gel should be completely
polymerized. - Clamp the apparatus (electrophoresis system), fill inner and
outer chamber with running buffer according to the manufac-
turer guidelines. - Load the samples and molecular mass marker into the wells for
separation by electrophoresis. - Run the gel at 50 V for 10 min, then run at 100 V for 70 min.
- After finishing the run, place the gel 1× transfer buffer for
10 min. - Assemble the transfer sandwich and make sure that there is no
air bubble in the transfer sandwich. - Place the transfer cassette to the trans-blot machine and trans-
fer the protein to the membrane followed the manufacturer
instruction of the instrument (see Note 8). - Rinse the PVDF membrane briefly with water and stain with
Ponceau S solution to check the transfer quality. - Wash the membrane with PBST to remove Ponceau S staining
(for three times). - Block in 5% dry milk for 1 h at room temperature.
- Incubate the membrane with MET primary antibody at 4 °C
overnight (see Note 9). - After incubation with primary antibody, wash the membrane
with PBST 10 min (for three times). - Incubate the blot with horse-reddish peroxide (HRP)-
conjugated secondary antibody for 1.5 h at room temperature
(see Note 10). - Wash the membrane with PBST for 10 min (three times).
Farhadul Islam et al.