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)
• Crude or enriched mouse liver mitochondria (See Preparation of Mitochondria from Mouse Liver)
• Formulation Buffer: 20 mM Tris (pH 8.5), 150 mM NaCl, 1 mM DTT, 20% Glycerol, 1 mg/mL fatty acid free BSA* (Sigma, Catalog # A6003)
• BID Dilution Buffer: 25 mM HEPES-KOH (pH 7.4), 0.1 M KCl, 1 mg/mL fatty acid free BSA*
• 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 recombinant human Bcl-w (MW: 19.6 kDa) in Formulation Buffer at 59 µg/mL. The final concentration will be 600 nM.
2. Prepare dilutions of recombinant human BIM-L (MW: 14.3 kDa) in Formulation Buffer at concentrations of 15,000, 5000, 1500, 500, 150, 50, and 15 nM. The final concentration range will be 3000 to 3 nM in a total reaction volume of 75 µL.
3. Aliquot 15 µL of each of the BIM-L dilutions to a series of tubes. Add 15 µL of recombinant human Bcl-w to each tube and gently mix. Incubate for 60 minutes at room temperature. Include one sample without recombinant human BIM-L (cleaved BID/Bcl-w control).
4. Prepare a stock solution of Caspase-8-cleaved recombinant human BID (MW: 22.0 kDa) in BID Dilution Buffer at 9.0 µg/mL. The final concentration will be 54 nM.
5. Add 10 µL of 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 BIM-L or Bcl-w (cleaved BID control).

   Initial BIM-L Conc.	BIM-L	Form.	rhBcl-w	Cleaved	BID Dil.	Mito	Mito	Final	Final
   	Vol.	Buffer	Vol.	BID	Buffer	Vol.	Buffer	Vol.	Conc.
   (nM)	(uL)	(uL)	(uL)	(uL)	(uL)	(uL)	(uL)	(uL)	(nM)
   15000	15	-	15	10	-	12	23	75	3000
   5000	15	-	15	10	-	12	23	75	1000
   1500	15	-	15	10	-	12	23	75	300
   500	15	-	15	10	-	12	23	75	100
   150	15	-	15	10	-	12	23	75	30
   50	15	-	15	10	-	12	23	75	10
   15	15	-	15	10	-	12	23	75	3
   Controls
   Cleaved BID + Bcl-w	-	15	15	10	-	12	23	75	0
   Cleaved BID only	-	30	-	10	-	12	23	75	0
   Spontaneous Release	-	30	-	-	10	12	23	75	0
   Total Cytochrome c	-	30	-	-	10	12	23	75	0

6. Add 12 µL of mitochondria (approximately 25-30 µg) and 23 µL of Mitochondria Buffer with added protease inhibitors and BSA to each tube.
7. Two additional 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.
8. Cap the tubes and gently mix the contents for 5-10 seconds. Incubate in a 30° C water bath for 30 minutes.
9. Total Cytochrome c in the assay should be determined by freezing the entire 75 µL reaction mix immediately after incubation at 30° C.
10. 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.
11. 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 Rat/Mouse Cytochrome c ELISA Kit product insert (Catalog # MCTC0).

Final Assay Conditions per 75 µL reaction

3000 to 3 nM recombinant human BIM-L
600 nM recombinant human Bcl-w
54 nM Caspase-8-cleaved recombinant human BID

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