Recombinant Human Bcl-2 Cytochrome c Release Assay

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)
• 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 1-5.

1. Prepare a stock solution of Caspase-8-cleaved recombinant human BID (MW: 22 kDa) in Dilution Buffer at 9.0 µg/mL. The final concentration will be 54 nM.
2. Prepare dilutions of recombinant human Bcl-2 (MW: 24.5 kDa) in Dilution Buffer at concentrations of 9000, 3000, 900, 300, 90, 30, and 9 nM. The final concentration range will be 3,000 to 3 nM in a 75 µL total reaction volume.
3. Aliquot 25 µL of each of the Bcl-2 dilutions to a series of tubes. Add 10 µL of the Caspase-8-cleaved recombinant human BID to each tube and gently mix. Incubate for 60 minutes at room temperature. Include one sample without recombinant human Bcl-2 (cleaved BID control).
4. Add 12 µL of mitochondria (approximately 25-30 µg) and 28 µL of Mitochondria Buffer with added 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.

   Initial Bcl-2 Conc.	Bcl-2	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
   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

6. Cap the tubes, and gently mix the contents for 5-10 seconds. Incubate in a 30° C water bath for 30 minutes.
7. Total Cytochrome c in the assay should be determined by freezing the entire 75 µL reaction mix immediately after incubation at 30° C.
8. 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.
9. Measure the levels of Cytochrome c in these samples using the Quantikine® Rat/Mouse Cytochrome c ELISA Kit (Catalog # MCTC0). See the Preparation of Samples for the Cytochrome c ELISA and additional instructions in the Quantikine Cytochrome c ELISA Kit product insert (Catalog # MCTC0).

Final concentration (per 75 µL reaction)

54 nM Caspase-8-cleaved recombinant human BID
3000 to 3 nM recombinant human Bcl-2

Quantikine is a registered trademark of R&D Systems.