Materials
- Recombinant mouse Bcl-xL (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)
- 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 stock solution should be prepared at 100 mg/mL.
Methods
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 1-6.
- Prepare a stock solution of Caspase-8-cleaved recombinant mouse BID (MW: 22 kDa) in Dilution Buffer at 9.0 µg/mL. The final concentration will be 54 nM.
- Prepare dilutions of recombinant mouse Bcl-xL (MW: 24.7 kDa) in Dilution Buffer at concentrations of 9000, 3000, 900, 300, 90, 30, 9, and 3 nM. The final concentration range will be 3000 to 1 nM in a total reaction volume of 75 µL.
- Aliquot 25 µL of each of the Bcl-xL dilutions to a series of tubes. Add 10 µL of the Caspase-8-cleaved recombinant mouse BID stock solution to each tube and gently mix. Incubate for 60 minutes at room temperature. Include one sample without recombinant mouse Bcl-xL (cleaved BID control).
- Add 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.
Initial Bcl-xL Conc. Bcl-xL Form. Cleaved Dil. Mito. Mito. Final Final Vol. Buffer BID Buffer Vol. Buffer Vol. Conc. (nM) (uL) (uL) (uL) (uL) (uL) (uL) (uL) (nM) 9000 25 - 10 - 12 28 75 3000 3000 25 - 10 - 12 28 75 1000 900 25 - 10 - 12 28 75 300 300 25 - 10 - 12 28 75 100 90 25 - 10 - 12 28 75 30 30 25 - 10 - 12 28 75 10 9 25 - 10 - 12 28 75 3 3 25 - 10 - 12 28 75 1 Controls Cleaved BID only - 25 10 - 12 28 75 0 Spontaneous Release - 25 - 10 12 28 75 0 Total Cytochrome c - 25 - 10 12 28 75 0 - 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.
- 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.
- Measure the levels of Cytochrome c in these samples using the mouse/rat Cytochrome c Quantikine® ELISA Kit (Catalog # MCTC0). See the Preparation of Samples for the Cytochrome c ELISA and additional instructions in the Cytochrome c Quantikine ELISA Kit product insert (Catalog # MCTC0).
Quantikine is a registered trademark of R&D Systems.