161- Electrode (running) buffer (10×): Dissolve 30 g of Tris base
(247 mM), 144 g glycine (1.92 M), and 10 g sodium dodecyl
sulfate (SDS) (0.1 % w/v) in 1 L of water, pH 8.3. Dilute to
1× running buffer in water prior to use. 1 L is suffi cient for one
electrophoretic run. - Prestained protein ladder ranging from 15 to 70 kDa (e.g.,
Fermentas PageRuler Prestained Protein Ladder, SM0671,
Thermo Scientifi c, or equivalent). - Nitrocellulose membrane, 0.45 μm.
- Western blot transfer buffer: 25 mM Tris base 192 mM gly-
cine, and 20 % methanol ( see Notes 5 and 6 ). - Mini-PROTEAN Tetra Cell and Mini Trans-Blot Module
(Bio- Rad) or equivalent equipment. - Power supply (10–300 V, 4–400 mA).
- 3MM Chr Blotting Paper (Whatman) or equivalent.
- Phosphate buffered saline (PBS; 10×): Dissolve 80 g of NaCl,
2 g of KCl, 14.4 g of Na 2 HPO 4 a, and 2.4 g of KH 2 PO 4 in
800 mL water. Make to 1 L with additional water. The pH in
the fi nal 1× PBS will become close to 7.4. - PBS containing 0.1–0.2 % Tween-20 (PBS-T).
- Blocking solution: 5 % dry nonfat milk in PBS ( see Note 7 ).
- Primary antibody dilution buffer: 1 % bovine serum albumin
(BSA), 0.1–0.2 % Tween-20 in PBS ( see Note 8 ). Store at 4 °C. - Enhanced Chemiluminescence (ECL) solution (GE Healtcare)
or equivalent. - Fuji medical X-ray fi lm super RX (Fujifi lm) or equivalent.
- Primary antibodies ( see Note 9 ):
Anti-caspase-2 mAb, clone 35 (BD Biosciences).
Anti-cleaved caspase-3 (Asp 175) pAb (Cell Signaling, Danvers).
Anti-cleaved caspase-6 pAb (Millipore, Billerica).
Anti-caspase-7 mAb, clone B94-1 (BD Biosciences).
Anti-caspase-8 mAb, clone 1C12 (Cell Signaling).
Anti-cleaved caspase-9 pAb (Cell Signaling).
Anti-glyceraldehyde-3-phosphate dehydrogenase (G3PDH)
pAb (e.g., from Trevigen). - Secondary antibodies:
HRP-conjugated anti-mouse IgG and anti-rabbit IgG (e.g.,
from Thermo Scientifi c). - Stripping buffer: 12.5 mL of 1 M Tris–HCl, pH 6.8, 770 μL
2-mercaptoethanol, 20 mL 10 % SDS. Make up to 100 mL
with water. Prepare immediately before use.
2.1.3 Western Blot
2.1.4 Immunodetection
Caspases in Mammalian Cell Cultures