Recombinant Mouse BID (Caspase-8-cleaved) Protein, CF Summary
Product Specifications
The EC50 for the desired application should be determined.
Uncleaved Recombinant Mouse BID is available (Catalog # 860-MB).
Met1-Asp195
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.
883-M8
| Formulation | Supplied as a 0.2 μm filtered solution in HEPES-KOH and KCl. |
| 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.
|
Assay Procedure
- Recombinant Mouse BID Caspase-8-cleaved (Catalog # 883-M8)
- Crude or enriched mouse liver mitochondria (See Preparation of mouse liver mitochondria at http://www.rndsystems.com/literature_cytochrome_c_release_assays_bcl-2.aspx)
- 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,(pH7.4), 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.
- Prepare dilutions of Recombinant Mouse BID Caspase-8-cleaved (MW: 22 kDa) in Dilution Buffer at concentrations of 500, 150, 50, 15, 5, 1.5, 0.5 and 0.15nM. The final concentration range will be 100 to 0.03 nM in a total reaction volume of 75 μL.
- Aliquot 15 μL of each of the BID dilutions to a series of tubes containg an additional 20 μL of Dilution Buffer and gently mix.
- Initiate the reaction by adding 12 μL of mitochondria (approximately 25-30 μg) and an additional 28 μL of Mitochondria Buffer with added protease inhibitors and BSA to each tube.
- 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.
- Cap the tubes and gently mix the contents for 5-10 seconds. Incubate in a 30 °C water bath for 30 minutes.
- 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 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-bcl2.aspx and additional instructions in the Rat/Mouse Cytochrome c Quantikine ELISA Kit product insert (Catalog # MCTC0).
Reconstitution Calculator
Background: BID
Bid is a 195 amino acid member of the Bcl‑2 family of proteins that regulates outer mitochondrial membrane permeability (1). Bid is a pro-apoptotic member that causes cytochrome c to be released from the mitochondria intermembrane space into the cytosol. In healthy cells Bid is cytosolic. In response to Fas ligand or TNF Bid is cleaved by Caspase-8 and it then relocates to the mitochondria outer membrane (2, 3). Cleavage of Bid by Caspase-8 generates a new N-terminal that contains a terminal glycine. It appears that the glycine is myristoylated and myristoylation serves to target Bid to the mitochondria (4). Bid may then interact with another pro-apoptotic Bcl‑2 family member Bak (5). Interaction of Bid with Bak causes altered mitochondrial membrane permeability. A 9-13 amino acid stretch called the BH3 region (Bcl‑2 homology region) appears to mediate the Bid interaction with other Bcl‑2 family members. Bid is neutralized by binding to the anti-apoptotic member Bcl‑xL.
- Gross, A. et al. (1999) Genes and Develop. 13:1899.
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Luo, X., et al. (1998) Cell 94:481.
- Li, H. et al. (1998) Cell 94:491.
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Zha, J. et al. (2000) Science 290:1761.
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Wei, M.C. et al. (2000) Genes Dev. 14:2060.
Citations for Recombinant Mouse BID (Caspase-8-cleaved) 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.
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Citations: Showing 1 - 4
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Anti-apoptotic BCL-2 proteins govern cellular outcome following B-RAF(V600E) inhibition and can be targeted to reduce resistance.
Authors: Serasinghe, M N, Missert, D J, Asciolla, J J, Podgrabinska, S, Wieder, S Y, Izadmehr, S, Belbin, G, Skobe, M, Chipuk, J E
Oncogene, 2014-03-10;34(7):857-67.
Species: Human
Sample Types: Whole Cells
Applications: Bioassay -
A novel cytotoxic sequence contributes to influenza A viral protein PB1-F2 pathogenicity and predisposition to secondary bacterial infection.
Authors: Alymova, Irina V, Samarasinghe, Amali, Vogel, Peter, Green, Amanda M, Weinlich, Ricardo, McCullers, Jonathan
J Virol, 2013-10-30;88(1):503-15.
Species: Mouse
Sample Types: Whole Cells
Applications: Bioassay -
Caspase-cleaved arrestin-2 and BID cooperatively facilitate cytochrome C release and cell death.
Authors: Kook, S, Zhan, X, Cleghorn, W M, Benovic, J L, Gurevich, V V, Gurevich, E V
Cell Death Differ, 2013-10-18;21(1):172-84.
Species: Mouse
Sample Types: Whole Cells
Applications: Bioassay -
Translocation of full-length Bid to mitochondria during anoikis.
Authors: Valentijn AJ, Gilmore AP
J. Biol. Chem., 2004-05-17;279(31):32848-57.
Applications: Western Blot
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