Dilution Buffer: 25 mM HEPES-KOH (pH 7.4), 0.1 M KCl, 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 stock solution should be prepared at 100 mg/mL.
Note: All buffers, proteins, and tubes should be kept on ice until indicated. Assay volumes are 75 µL and are combined in 0.5 mL Eppendorf tubes. The table below Step 5 summarizes the reactions that should be set up in Steps 1-5.
Prepare dilutions of Caspase-8-cleaved recombinant human BID (MW: 22 kDa) in Dilution Buffer at concentrations of 500, 150, 50, 15, 5, 1.5, 0.5, and 0.15 nM. The final concentration range will be 100 to 0.03 nM in a total reaction volume of 75 µL.
Aliquot 15 µL of each of the BID dilutions to a series of tubes containing an additional 20 µL of Dilution Buffer and gently mix.
Initiate the reaction by adding 12 µL of mitochondria (approximately 25-30 µg) and 28 µL of Mitochondria Buffer containing protease inhibitors and BSA to each tube.
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.
Cap the tubes and gently mix the contents for 5-10 seconds. Incubate in a 30 °C water bath for 30 minutes.
Total Cytochrome c in the assay should be determined by freezing the entire 75 µL reaction mix immediately after incubation at 30 °C.
Centrifuge the remaining samples at 16,000 x g for 5 minutes 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.