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 280

2. Materials

2.1. Cell Culture
and Treatments

1. RPMI 1640 Medium (Sigma-Aldrich) supplemented with 10%
fetal bovine serum (HyClone Laboratories, Logan, UT), peni-
cillin (100 U/ml), and streptomycin (100 mg/ml) (HyClone).
2. T-75 tissue culture flasks (Sarstedt, Nümbrecht, Germany).
3. Peroxynitrite was synthesized by the reaction of nitrite with
acidified H 2 O 2 , as described previously ( 12 ), with minor
modifications ( 13 ) (see Note 1).
4. Saline A: 8.182 g/l NaCl, 0.372 g/l KCl, 0.336 g/l NaHCO 3,
and 0.9 g/l glucose, pH 7.4 at 37°C.

2.2. Cell Fractionation

1. Extraction buffer: 20 mM HEPES-KCl (pH 7.4), 10 mM
KCl, 250 mM sucrose, 1.5 mM MgCl 2 , 1 mM sodium EDTA,
1 mM sodium EGTA. Immediately before use add 1 mM
dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride
(PMSF), 10 mg/ml leupeptin, 10 mg/ml pepstatin, and
10 mg/ml aprotinin.
2. Lysis buffer: 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1%
NP40. Immediately before use add 1 mM DTT, 1 mM PMSF,
10 mM sodium orthovanadate (Na 3 VO 4 ), 10 mM sodium
fluoride (NaF), 10 mg/ml leupeptin, 10 mg/ml pepstatin,
and 10 mg/ml aprotinin.

2.3. SDS-
Polyacrylamide Gel
Electrophoresis

1. Bio-Rad Dye Binding protein assay (Bio-Rad Laboratories,
Hercules, CA).
2. Mini gel electrophoresis apparatus (e.g., BioRad Mini-
PROTEAN 3 electrophoresis system).
3. Loading buffer: 125 mM Tris-HCl (see Note 2), 2% (w/v)
sodium dodecyl sulphate (SDS), 10% glycerol, 0.002% bro-
mophenol blue, pH 6.9. Prepare a 4× solution and store in
aliquots at −20°C. Before use add 100 mM DTT.
4. Upper Tris (4×): 0.5 M Tris-HCl, pH 6.8. Store at 4°C (see
Note 2).
5. Lower Tris (4×): 1.5 M Tris-HCl, pH 8.8. Store at 4°C (see
Note 2).
6. Forty percent acrylamide/bisacrylamide solution: 39:1 w/w;
this is a neurotoxin when unpolymerized and so care should
be taken not to receive exposure (see Note 3).
7. Ammonium persulfate (APS): prepare a 10% solution in water
(see Note 4).
8. Stacking gel solution (4%): prepare 100 ml of solution by
mixing 64 ml of water, 10 ml of 40% acrylamide, 25 ml of