Biology Reference
In-Depth Information
Microdomains
22.4.2.2
Performing blue-native gel electrophoresis
Prepare 6% and 15% resolving gel mixture according to
Table 22.1
.
Fill the tubing and 1 cm of cassette height with ddH
2
O.
Fill the front reservoir of the gradient mixer with 2.8 mL of 6% gel mixture and
the rear reservoir with 2.3 mL of 15% gel mixture and then place the conical
insert into the rear reservoir.
Fill the gel cassette by means of underlaying the gel under water using a
peristaltic pump at speed 9.
After polymerization, wash gel surface with 1
gel buffer.
Prepare 2.5 mL of 4% stacking gel mixture according to
Table 22.1
, then insert
comb,
and fill to the top with stacking gel mixture.
After polymerization, remove comb and wash wells with 1
GB.
Fill the wells with blue cathode buffer and underlay the samples into the wells
(10-30
g protein per well).
Place gel cassettes into the apparatus and fill upper and lower reservoir with blue
cathode buffer and anode buffer, respectively.
Run for 15 min at 40 V and then at 80 V until the dye reaches 2/3 of the gel.
Replace the blue cathode buffer with cathode buffer and continue the
electrophoresis until the dye front reaches the end of the gel.
Remove the gel from the plates and proceed with either (i) Western blotting, (ii)
2D gel electrophoresis, or (iii) silver staining (
Fig. 22.1
).
m
22.4.2.3
Analysis by Western blotting
For Western blotting, place gels in Towbin semidry transfer buffer for 20 min on
a rocker.
Place a polyvinylidene fluoride (PVDF) membrane in methanol for 1 min, rinse
three times with ddH
2
O, and then place in Towbin semidry transfer buffer for
20 min on a shaker.
Table 22.1
Preparation of Gradient Gels for Blue-Native Gel Electrophoresis
4% Stacking Gel
6% Resolving Gel
15% Resolving Gel
2.5 mL
5.0 mL
2.5 mL
5.0 mL
2.5 mL
5.0 mL
ddH
2
O
1.45 mL
2.87 mL
2.72 mL
5.44 mL
0.84 mL
1.68 mL
3 Gel buffer
0.82 mL
1.64 mL
1.65 mL
3.3 mL
1.65 mL
3.3 mL
Acrylamide/
bisacrylamide
0.2 mL
0.4 mL
0.6 mL
1.2 mL
1.5 mL
3.0 mL
Glycerol
-
-
-
-
1 mL
2 mL
Ammonium
persulfate
30
m
L
60
m
L
30
m
L
60
m
L
5
m
L
10
m
L
TEMED
3
m
L
6
m
L
2
m
L
4
m
L
2
m
L
4
m
L
Volumes listed are sufficient for 1 or 2 gels (i.e., 2.5 or 5.0 mL, respectively).
