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 281

Upper Tris, 1 ml of 10% SDS. Store at 4°C. Add 4 ml of
N,N,N,N ′-Tetramethyl-ethylendiamide (TEMED, Sigma-
Aldrich) (see Note 5) and 40 ml of APS to 4 ml of the stacking
gel solution.
9. Running gel solution (12.5%): prepare 10 ml of solution by
mixing 4.3 ml of water, 3.1 ml of 40% acrylamide, 2.5 ml of
Lower Tris, 100 ml of 10% SDS, 14 ml of TEMED and 60 ml
of APS.
10. Pre-stained molecular weight markers: Color Burst (Sigma-
Aldrich).

2.4. Western Blotting
for Mitochondrial
and Cytosolic
Fractions

1. Blotting apparatus (e.g., Bio-Rad Mini-PROTEAN 3 full
immersion electroblotting system).
2. Electrophoresis Buffer (10×): 250 mM Tris (pH 8.3), 1.92 M
glycine, 0.1% SDS. Store at room temperature.
3. Transfer Buffer (10×): 250 mM Tris (pH 8.3), 1.92 M gly-
cine. Store at room temperature, dilute 1:10 in water and add
20% (v/v) methanol prior to use (see Note 6).
4. Polyvinyldiene (PVDF) membrane (GE Healthcare).
5. Tris-buffered saline with Tween (TBS-T): prepare a 10× solu-
tion with 1.40 M NaCl, 500 mM Tris-HCl, pH 7.2. Dilute
100 ml with 900 ml water for use and complete with 0.1%
Tween-20 (see Note 7).
6. Blocking buffer: 5% (w/v) nonfat dry milk in TBS-T.
7. Primary antibody dilution buffer: dilute Bad primary anti-
body (BD Transduction Laboratories, Lexington, KY) 1:700
in 4 ml of blocking buffer (see Note 8). Dilute anti-actin and
anti-HSP-60 antibodies (Santa Cruz, Santa Cruz, CA)
1:1,000 in 4 ml of blocking buffer.
8. Secondary antibody: dilute antimouse IgG conjugate to horse
radish peroxidase (Santa Cruz) 1:2,000 in 4 ml of blocking
buffer.
9. Enhanced chemiluminescent (ECL) reagent (Sigma-Aldrich):
mix 10 ml of solution A (1 M Tris-HCl, pH 8.6, 1.25 mM
luminol) with 3.2 ml of 30% H 2 O 2 and 50 ml of solution B
(6.7 mM p-Coumaric acid in DMSO).
10. X-ray film and exposure cassette.

3. Methods

A significant amount of Bad is normally detected in the mito-
chondria of untreated U937 cells. Exposure of these cells to a
non-toxic, but nevertheless MPT-committing concentration of