Assessing Bad Sub-cellular Localization Under Conditions Associated with Prevention or Promotion of Mitochondrial Permeability Transition-Dependent Toxicity - Protein Misfolding and Cellular Stress in Disease and Aging - page 285

3. The gel unit is disconnected from the power supply and
disassembled. Remove the stacking gel and soak (30 s) the
remaining portion of the gel in transfer buffer. Place the gel
on top of the blotting-paper and make sure that it remains
submerged in the transfer buffer.
4. Soak a PVDF membrane (approximately 6 × 9 cm, same size
as the running gel) in 100% methanol (1 min). The mem-
brane should then be rinsed with the transfer buffer (1-2 min)
and equilibrated in the same buffer for at least 30 min.
Finally, place the membrane on top of the gel in the transfer
cassette.
5. Carefully lay three sheets of blotting-paper and a sponge
(pre-wetted with the transfer buffer) on top of the PVDF
membrane, ensuring that no bubbles are trapped in the
resulting sandwich. Close the transfer cassette.
6. Place the cassette into the transfer tank. In order to prevent
loss of the proteins into the transfer buffer, the PVDF mem-
brane should be between the gel and the anode.
7. Insert the Bio-Ice cooling unit in the transfer tank. This is
necessary to optimize protein transfer and to prevent damage
to the electroblotting system due to heat generated during
transfer.
8. Fill the transfer tank with the transfer buffer.
9. Complete the assembly of the electroblotting system and
connect to a power supply (36 V for 75 min).
10. After protein transfer, the transfer cassette should be removed
from the tank and immediately disassembled. Remove the
upper sponge/blotting-paper sheets and lay the PVDF
membrane on clean paper. The gel and blotting-paper can be
discarded. Mark positions of molecular weight markers with a
pen (see Note 14).
11. The membrane is then incubated for 1 h (at room tempera-
ture) in 20 ml of blocking buffer (Subheading 2.4 , point 6) on
a rocking platform to prevent nonspecific protein binding.
12. Discard the blocking buffer and quickly rinse the membrane
prior to addition of the anti-Bad antibody (diluted as detailed
in Subheading 2.4 , point 7). The membrane is then incu-
bated overnight (4°C) on a roller mixer.
13. After overnight incubation, wash the membrane with 20 ml
TBS-T at room temperature (1 × quick rinse, 1 × 15 min and
2 × 10 min).
14. The secondary antibody should be freshly prepared
(Subheading 2.4 , point 8) in each experiment. Add the anti-
body-containing solution to the membrane and allow incuba-
tion for 90 min (at room temperature) on a rocking platform.