Recombinant Human Bcl-2 related protein A1 (aa 1-152), CF

• Recombinant Human Bcl‑2 related protein A1 Minus C-terminus (Recombinant Human BCL2A1) (Catalog # 1160-A1)
• Recombinant Human BID Caspase-8-cleaved (Catalog # 882-B8)
• Crude or enriched mouse liver mitochondria (See Preparation of mouse liver mitochondria at http://www.rndsystems.com/literature_cytochrome_c_release_assays_bcl-2.aspx)
• Dilution Buffer: 25 mM HEPES-KOH (pH 7.4), 0.1 M KCl, 10% Glycerol, 1 mg/mL fatty acid free BSA* (Sigma, Catalog # A6003) 
• Mitochondria Buffer: 125 mM KCl, 0.5 mM MgCl₂, 3.0 mM Succinic acid, 3.0 mM Glutamic acid, 10 mM HEPES-KOH (pH 7.4), 1 mg/mL BSA*, containing 25 μg/mL Leupeptin, 25 μg/mL Pepstatin, 3 μg/mL Aprotinin, 100 μM PMSF, and 10 μM Boc-Asp-FMK caspase inhibitor
  *Note: Protease inhibitors and BSA should be added to the buffer immediately prior to use. BSA solution should be prepared at 100 mg/mL.

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1. Prepare a stock solution of Recombinant Human BID Caspase-8-cleaved (MW: 22 kDa) in Dilution Buffer at 9.0 μg/mL. The final concentration will be 54 nM.
2. Prepare dilutions of Recombinant Human BCL2A1 (MW: 18.3 kDa) in Dilution Buffer at concentrations of 9000, 3000, 900, 300, 90, 30, 9 and 3 nM.  The final concentration range will be 3,000 to 1 nM in a total rxn volume of 75 μL.
3. Aliquot 25 μL of each of the BCL2A1 dilutions to a series of tubes. Add 10 μL of the Recombinant Human BID Caspase-8-cleaved to each tube and gently mix. Incubate for 60 min. at room temperature. Include one sample without Recombinant Human BCL2A1 (cleaved BID control).
4. Add 12 μL of mitochondria (approximately 25-30 μg) and 28 μL of Mitochondria Buffer containing protease inhibitors and BSA to each tube.
5. Two control samples must be run for each assay to determine the total amount of Cytochrome c that can be released from the mitochondria and the amount of spontaneously released Cytochrome c. Set up two samples containing only mitochondria and the appropriate buffers that have not been treated with any test proteins.
6. Cap the tubes and gently mix the contents for 5-10 seconds. Incubate in a 30 °C water bath for 30 min.
7. Total Cytochrome c in the assay should be determined by freezing the entire 75 μL rxn mix immediately after incubation at 30 °C.
8. Centrifuge the remaining samples at 16,000 x g for 5 min. at 2-8 °C. Remove and transfer a 50 μL aliquot of the supernatant to a new chilled tube. Samples may be analyzed immediately or stored at -20 °C in a manual defrost freezer.
9. Measure the levels of Cytochrome c in these samples using the Rat/Mouse Cytochrome c Quantikine® ELISA Kit (Catalog # MCTC0). See the Preparation of Samples for the Cytochrome c ELISA at http://www.rndsystems.com/literature_cytochrome_c_release_assays_bcl-2.aspx and additional instructions in the Rat/Mouse Cytochrome c Quantikine ELISA Kit product insert (Catalog # MCTC0).