Recombinant Human BID Protein, CF
Recombinant Human BID Protein, CF Summary
Cytochrome c is quantified using the Rat/Mouse Cytochrome c Quantikine® ELISA Kit (Catalog # MCTC0).
The EC50 for the desired application should be determined.
Recombinant Human BID Caspase-8-cleaved is available (Catalog # 882-B8).
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.
|Formulation||Supplied as a 0.2 μm filtered solution in HEPES 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.
- Recombinant Human BID (Catalog # 846-BD)
- 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)
- Dilution Buffer: 25 mM HEPES-KOH (pH 7.4), 0.1 M KCl, 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 solution should be prepared at 100 mg/mL.
- Prepare dilutions of Recombinant Human BID (MW: 22 kDa) in Dilution Buffer at concentrations of 5000, 1500, 500, 150, 50, 15, 5 and 1.5 nM. The final concentration range will be 1000 to 0.3 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 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 minutes.
- 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).
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-terminous 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 withanother 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.
- Luo, X., et al. (1998) Cell 94:481.
- Li, H. et al. (1998) Cell 94:491.
Zha, J. et al. (2000) Science 290:1761.
Wei, M.C. et al. (2000) Genes Dev. 14:2060.
Citations for Recombinant Human BID 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.
Citations: Showing 1 - 2
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Identification of a novel protein MICS1 that is involved in maintenance of mitochondrial morphology and apoptotic release of cytochrome c.
Authors: Oka T, Sayano T, Tamai S, Yokota S, Kato H, Fujii G, Mihara K
Mol. Biol. Cell, 2008;19(6):2597-608.
Sample Types: Whole Cells
Acyl coenzyme A-binding protein augments bid-induced mitochondrial damage and cell death by activating mu-calpain.
Authors: Shulga N, Pastorino JG
J. Biol. Chem., 2006;281(41):30824-33.
Sample Types: Cell Lysates
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Quantikine Colorimetric Sandwich ELISAs
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