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 284

rapid polymerization) to prepare a 2-3 mm thick border on
the bottom of the gel cassette. Complete the running gel
with the above gel solution (approximately 9.5 ml) and APS
(60 µl) and immediately pour into the gel cassette (6 cm from
the bottom). Overlay with isobutanol (about 700-800 µl)
and allow polymerization for about 30 min.
3. Pour off the isobutanol layer, rinse twice the top of the gel
with water and then carefully dry with a thin blotting-paper.
4. Add to the stacking gel solution (4 ml), prepared as detailed
above (Subheading 2.3 , point 8), 4 ml of TEMED and 40 ml
of APS. Immediately pour this solution on top of the running
gel. Insert the comb and allow polymerization for about
30 min.
5. Prepare the electrophoresis buffer by diluting 100 ml of the
10× electrophoresis buffer (Subheading 2.4 , point 2) with
900 ml of water.
6. Electrophoresis was performed with a Bio-Rad Mini-
PROTEAN 3 electrophoresis gel system. Place the gel cassette
sandwich into the electrode assembly and then in the inner
chamber of the electrophoresis module. The inner chamber is
finally lowered into the mini tank.
7. Fill the inner chamber with the electrophoresis buffer and
subsequently add the same solution in the mini tank (3 cm
from the bottom). Carefully remove the comb and use a 50 ml
syringe, fitted with a 22-gauge needle (see Note 13), to rinse
the wells with the electrophoresis buffer.
8. Load each sample in a well with the same 50 µl syringe.
Carefully rinse the syringe with electophoresis buffer when
loading the different samples. Load one well with pre-stained
molecular weight markers. The first and the last wells should
be loaded with the loading buffer alone.
9. Complete the assembly of the gel unit and connect to a power
supply. The gel is generally run at 55 V through the stacking
gel and at 110-120 V through the running gel. In order to
prevent loss of low molecular weight proteins, electrophore-
sis should be stopped immediately prior to or soon after the
dye front runs off the gel. The dye front is blue because of the
presence of bromophenol blue.

3.3. Western Blotting
for Detection
of Mitochondrial
and Cytosolic Bad

1. After SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE),
proteins are transferred to PVDF membranes using a Bio-Rad
Mini-PROTEAN 3 full immersion electroblotting system.
2. Place the transfer cassette in a tray and add enough transfer
buffer (Subheading 2.4 , point 3) to submerge the lower side
of the cassette, in which a piece of sponge sheet and three
sheets of blotting-paper are sequentially layered.