Apoptosis Assays

Recombinant Mouse Caspase-8-cleaved BID Cytochrome c Release Assay

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)

Preparation of the Samples for Cytochrome c ELISA

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.

Recombinant Mouse BID Cytochrome c Release Assay

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)

Recombinant Mouse Bcl-x

Materials

Recombinant Human Caspase-8-cleaved BID Cytochrome c Release Assay

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)

Recombinant Human BIM-L Cytochrome c Release Assay

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)

Recombinant Human BID Cytochrome c Release Assay

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)

Recombinant Human Bcl-x

Materials

Caspase Activity Assays for Inhibitors of Apoptosis & Mitochondrial Proteins

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.

Recombinant Mouse Bcl-xL Cytochrome c Release Assay

Materials

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