>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
Measured by its ability to inhibit Caspase-8-cleaved BID-mediated release of cytochrome c from isolated mouse liver mitochondria using the Rat/Mouse Cytochrome c Quantikine ELISA (Catalog # MCTC0) to quantify cytochrome c. The typical EC50 for Bcl-xL in this assay is <400 nM in the presence 54 nM of Recombinant Human or Mouse BID Caspase-8-cleaved (Catalog # 882-B8 or 883-M8, respectively). Optimal dilutions should be determined by each laboratory for each application.
E. coli-derived mouse Bcl-xL protein Ser2-Arg212, with a C-terminal 6-His tag
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.
Note: All buffers, proteins and tubes should be kept on ice. Assay volumes are 75 μL and are combined in 0.5 mL Eppendorf tubes.
Prepare a stock solution of Recombinant Mouse BID Caspase-8-cleaved (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 Minus C‑Terminus (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 3,000 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 Recombinant Mouse BID Caspase-8-cleaved to each tube and gently mix. Incubate for 60 min. at room temperature. Include one sample without Recombinant Mouse Bcl-xL Minus C‑Terminus (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 min.
Total Cytochrome c in the assay should be determined by freezing the entire 75 μL rxn mix immediately after incubation at 30 °C.
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.
Bcl-xL is a member of the Bcl-2 family of proteins that regulates outer mitochondrial membrane permeability (1, 2). Bcl-xL is an anti‑apoptotic member that prevents release of cytochrome c from the mitochondria intermembrane space into the cytosol. Bcl-xL is present on the outer mitochondrial membrane and is also found on other membranes in some cell types. Natural Bcl-xL contains a carboxyl‑terminal mitochondria targeting sequence. Recombinant Bcl-xL missing the mitochondrial targeting sequence maintains its ability to neutralize pro‑apoptotic Bcl-2 family members. Neutralization by Bcl-xL appears to be through binding the BH3 region of pro‑apoptotic Bcl-2 family members. This activity does not require the mitochondrial targeting sequence.
Gross, A. et al. (1999) Genes and Develop. 13:1899.
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