Apoptosis Assays

Materials

• Caspase-8-cleaved recombinant mouse BID (R&D Systems Catalog # 883-M8)
• Crude or enriched mouse liver mitochondria (See Preparation of Mitochondria from Mouse Liver)
• Dilution Buffer: 25 mM HEPES-KOH (pH 7.4), 0.1 M KCl, 1 mg/mL fatty acid free BSA* (Sigma Catalog # A6003)

Materials

• Quantikine® Rat/Mouse Cytochrome c ELISA Kit (R&D Systems Catalog # MCTC0)
• 5% Triton® X-100

Methods

1. Thaw the samples immediately prior to running the ELISA.
2. Add 7.5 µL of 5% Triton® X-100 to the total Cytochrome c control sample (75 µL) that was frozen immediately after incubation at 30° C.

Materials

• Recombinant mouse BID (R&D Systems Catalog # 860-M8)
• Crude or enriched mouse liver mitochondria (See Preparation of Mitochondria from Mouse Liver)
• Dilution Buffer: 25 mM HEPES-KOH (pH 7.4), 0.1 M KCl, 1 mg/mL fatty acid free BSA* (Sigma Catalog # A6003)

Materials

• Caspase-8-cleaved recombinant human BID (R&D Systems Catalog # 882-B8)
• Crude or enriched mouse liver mitochondria (See Preparation of Mitochondria from Mouse Liver)
• Dilution Buffer: 25 mM HEPES-KOH (pH 7.4), 0.1 M KCl, 1 mg/mL fatty acid free BSA* (Sigma Catalog # A6003)

Materials

• Recombinant human BIM-L (R&D Systems Catalog # 1325-BL)
• Caspase-8-cleaved recombinant human BID (R&D Systems Catalog # 882-B8)
• Recombinant human Bcl-w (R&D Systems Catalog # 824-BW)

Materials

• Recombinant human BID (R&D Systems Catalog # 846-BD)
• Crude or enriched mouse liver mitochondria (See Preparation of Mitochondria from Mouse Liver)
• Dilution Buffer: 25 mM HEPES-KOH (pH 7.4), 0.1 M KCl, 1 mg/mL fatty acid free BSA* (Sigma Catalog # A6003)

Materials

• Recombinant human Bcl-x_L (R&D Systems Catalog # 894-BX)
• Caspase-8-cleaved recombinant human BID (R&D Systems Catalog # 882-B8)
• Crude or enriched mouse liver mitochondria (See Preparation of Mitochondria from Mouse Liver)

As their name implies, a primary role for members of the Inhibitors of Apoptosis (IAP) family lies in their ability to suppress apoptosis. This occurs through the inhibition of pro-apoptotic members of the Caspase family. IAP proteins are characterized structurally by the presence of at least one BIR (baculoviral IAP repeat) domain. Binding of the BIR domain of IAPs to active caspases inhibits caspase activity. Eight IAPs have been identified in humans including Apollon/BRUCE, cIAP-1, cIAP-2, ILP-2, Livin, NAIP, Survivin, and XIAP.

Materials

• Recombinant mouse Bcl-x_L (R&D Systems Catalog # 878-BC)
• Caspase-8-cleaved recombinant mouse BID (R&D Systems Catalog # 883-M8)
• Crude or enriched mouse liver mitochondria (See Preparation of Mitochondria from Mouse Liver)

Materials

• Recombinant human Bcl-2 (R&D Systems Catalog # 827-BC)
• Caspase-8-cleaved recombinant human BID (R&D Systems Catalog # 882-B8)
• Crude or enriched mouse liver mitochondria (See Preparation of Mitochondria from Mouse Liver)