Biomedical Engineering Reference
In-Depth Information
4. Initiate accumulation by addition of a tenfold excess of
MTX-coupled precursor (240 ng, corresponding to 6.25 pmol
in the case of b
2
(167)
ʔ
-DHFR), and incubate for 15 min
at 25 °C.
5. Stop reaction by addition of 1× inhibitor mix, and move sam-
ples on ice.
6. Pellet mitochondria by centrifugation at 12,000 ×
g
for 10 min.
7. Wash mitochondria by addition of 200
ʼ
l SEM buffer and cen-
trifugation at 12,000 ×
g
for 8 min.
8. Solubilize mitochondrial pellet in 55
l ice-cold digitonin buf-
fer by pipetting up and down carefully with a 20- 200
ʼ
µl-pipet-tip
for 15-20 times without causing digitonin foam. Keep on ice for
15 min.
9. Spin down unsolubilized material by centrifugation at
20,000 ×
g
for 15 min.
10. Prepare fresh tubes with 5
-
l
of solubilized material (supernatants) is added after centrifuga-
tion. Take 5
ʼ
l 10× Loading Dye, to which 45
ʼ
l of remaining supernatants each, and analyze on
SDS-PAGE in parallel.
11. Load samples on a 6-16.5 % acrylamide gradient BN gel (
see
Table
1
and
Note 10
). Cover samples with 1× cathode buffer
containing 0.2 % coomassie blue G250. Gel run is performed
in a cooled chamber (4 °C). Keep at 100 V for passage through
the stack gel, afterwards the voltage can be raised to
600 V. Always keep the current below 15 mA.
12. After the proteins have migrated into the separation gel, the
Coomassie blue-containing cathode buffer is replaced by the
one without dye (excess dye might interfere with subsequent
Western transfer of the proteins).
13. Before Western transfer, the gel is incubated in 1x SDS-PAGE
running buffer for 5 min (facilitation of protein transfer).
ʼ
Table 1
Pipetting scheme for blue native gel preparation
% Acrylamide
6
8
10
13
16.5
Stack gel
3× gel buffer (ml)
3
3
3
3
3
2.5
Acrylamide (ml)
1.07
1.46
1.82
2.35
3.05
0.6
Glycerol (ml)
-
-
1.8
1.8
1.8
-
Water (ml)
4.888 4.507 2.347 1.817 1.117 4.367
10 % APS (
ʼ
l)
38
38
38
38
38
30
TEMED (
ʼ
l)
3.8
3.8
3.8
3.8
3.8
3
