Recombinant Human Bcl-w (minus C-Terminus) Protein, CF Summary
Product Specifications
Optimal dilutions should be determined by each laboratory for each application.
Ala2-Thr172, with a C-terminal 6-His tag
Analysis
Product Datasheets
Carrier Free
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
824-BW
| Formulation | Supplied as a 0.2 μm filtered solution in HEPES, NaCl and Glycerol. |
| Shipping | The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage: | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
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Assay Procedure
- Recombinant Human Bcl‑w Minus C‑Terminus (Catalog # 824-BW)
- Recombinant Human BID Caspase-8-cleaved (Catalog # 882-B8)
- Crude or enriched mouse liver mitochondria (http://www.rndsystems.com/literature_cytochrome_c_release_assays_bcl-2.aspx)
- Dilution Buffer: 25 mM HEPES-KOH (pH 7.4), 0.1 M KCl, 10% Glycerol, 1 mg/mL fatty acid free BSA* (Sigma, Cat. # 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 solution should be prepared at 100 mg/mL.
- Prepare a stock solution of Recombinant Human 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 Human Bcl‑w Minus C‑Terminus (MW: 19.5 kDa) in Dilution Buffer at concentrations of 30, 9, 3, 0.9, 0.3, 0.09 and 0.03 nM. The final concentration range will be 10 to 0.01 nM in a total rxn volume of 75 μL.
- Aliquot 25 μL of each of the Bcl-w dilutions to a series of tubes. Add 10 μL of the Recombinant Human BID Caspase-8-cleaved to each tube and gently mix. Incubate for 60 min. at room temperature. Include one sample without Recombinant Human Bcl‑w 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.
- Measure the levels of Cytochrome c in these samples using the Rat/Mouse Cytochrome c Quantikine® ELISA Kit (Catalog # MCTC0). See the Preparation of Samples for the Cytochrome c ELISA at http://www.rndsystems.com/literature_cytochrome_c_release_assays_bcl-2.aspx and additional instructions in the Rat/Mouse Cytochrome c Quantikine ELISA Kit product insert (Catalog # MCTC0).
Reconstitution Calculator
Background: Bcl-w
Bcl-w (also known as Bcl-2 like protein 2, BCL2L2) is a member of the Bcl-2 family of proteins that regulates outer mitochondrial membrane permeability (1, 2). Bcl-w is an anti-apoptotic member that prevents release of cytochrome c from the mitochondria intermembrane space into the cytosol. Bcl-w is required for normal sperm maturation (3, 4, 5). Natural Bcl-w contains a carboxyl-terminal mitochondria targeting sequence. Recombinant Bcl-w missing the mitochondrial targeting sequence maintains its ability to neutralize pro-apoptotic Bcl-2 family members. Neutralization by Bcl-w 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.
- Kroemer, G. (1997) Nature Med. 3:614.
- Ross, J.A., et al. (1998) Nat. Genet. 18:251.
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Print, C.G. et al. (1998) Proc. Natl. Acad. Sci. USA 95:12323.
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Yan, W. et al. (2000) Mol. Endocrin. 14:682.
Citation for Recombinant Human Bcl-w (minus C-Terminus) Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
1 Citation: Showing 1 - 1
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Target-derived neurotrophins coordinate transcription and transport of bclw to prevent axonal degeneration.
Authors: Cosker, Katharin, Pazyra-Murphy, Maria F, Fenstermacher, Sara J, Segal, Rosalind
J Neurosci, 2013-03-20;33(12):5195-207.
Species: Mouse
Sample Types: Whole Cells
Applications: Bioassay
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