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 286

15. Discard the secondary antibody and wash the membrane
three times (10 min each) with 20 ml of TBS-T.
16. Soak the membrane in ECL reagent under safe light condi-
tions. Rotate the membrane by hand for 1 min (to ensure
even coverage), remove the excess ECL reagent with blotting
paper and immediately place the membrane between the
leaves of an acetate sheet protector (cut to the size of an X-ray
film cassette) (see Note 15).
17. The final step is performed in the dark room. Place the mem-
brane-containing acetate sheet in an X-ray film cassette. Place
the film on top of the membrane for a suitable exposure time,
typically 10 min. This time can be decreased or increased,
depending on the results obtained.

3.4. Re-probing Blots
for Actin and HSP-60

1. After film exposure, strip the membrane of the Bad signal and
re-probe with anti-actin or anti-HSP-60 antibodies to assess
equal loading of the lanes and the purity of the mitochondrial
fractions.
2. Since the above proteins display remarkably different molecu-
lar weights, the stripping procedure is simplified as detailed
below.
3. Wash the membrane with TBS-T, for 1 h, on a rocking plat-
form with occasional change of buffer. The membrane is now
ready to be re-probed with anti-actin or anti-HSP-60 (diluted
as detailed in Subheading 2.4 , point 7) antibodies, using the
same procedure described for the anti-Bad antibody.

4. Notes

1. Solutions should be prepared in distilled or deionized water.
2. Prepare the Tris buffer by dissolving the Tris base in water
and adjusting the pH with HCl. Adjust to pH 6.8 for Upper
Tris and to pH 8.8 for Lower Tris. Tris base cannot be
replaced by Tris-Cl, or Trizma, as the salt concentration
would be too high and, under these conditions, polypeptides
migrate anomalously through the gel, yielding extremely
diffuse bands.
3. In solution, acrylamide and bisacrylamide are slowly con-
verted during storage to acrylic acid and bisacrylic acid. These
reactions are catalyzed by light or alkali. Check that the pH of
the solution is 7.0 or less. Store the solution in dark bottles.
Fresh solutions should be prepared every few months.
4. APS provides the free radicals that drive polymerization of
acrylamide and bisacrylamide. A small amount of a 10% (w/v)