Recombinant Human BID Protein, CF

R&D Systems | Catalog # 846-BD

R&D Systems
Discontinued Product
846-BD has been discontinued. View all BID products.

Key Product Details

Source

E. coli

Accession Number

P55957

Applications

Bioactivity
View all BID Proteins and Enzymes »

Product Specifications

Source

E. coli-derived human BID protein
Met1-Asp195

Purity

>95%, by SDS-PAGE under reducing conditions and visualized by silver stain.

Endotoxin Level

<0.01 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

Met1

Predicted Molecular Mass

22 kDa

SDS-PAGE

22 kDa, under reducing and non-reducing conditions

Activity

Measured by its ability to induce cytochrome c release from isolated mouse liver mitochondria.
The typical EC50 for cytochrome c releasing activity is less than 200 nM.
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).

Formulation, Preparation, and Storage

846-BD
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.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.

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-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.

 

References

  1. Gross, A. et al. (1999) Genes and Develop. 13:1899.
  2. Luo, X., et al. (1998) Cell 94:481.
  3. Li, H. et al. (1998) Cell 94:491.
  4. Zha, J. et al. (2000) Science 290:1761.
  5. Wei, M.C. et al. (2000) Genes Dev. 14:2060.

Long Name

BH3 Interacting Domain Death Agonist

Alternate Names

apoptic death agonist, BH3 interacting domain death agonist, BH3-interacting domain death agonist, BID isoform ES(1b), BID isoform L(2), BID isoform Si6, desmocollin type 4, FP497, Human BID coding sequence, MGC15319, MGC42355, p22 BID, tbid

Entrez Gene IDs

637 (Human); 12122 (Mouse)

Gene Symbol

BID

UniProt

P55957

Additional BID Products

Product Documents for Recombinant Human BID Protein, CF

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Recombinant Human BID Protein, CF

For research use only

Related Research Areas

Bcl-2 Proteins and Regulators

Citations for Recombinant Human BID Protein, CF

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Protocols

View specific protocols for Recombinant Human BID Protein, CF (846-BD):

Materials
  • 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.
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

.

  1. 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.
  2. 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. 
  3. 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.
  4. 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.
  5. Cap the tubes and gently mix the contents for 5-10 seconds. Incubate in a 30 °C water bath for 30 minutes.
  6. Total Cytochrome c in the assay should be determined by freezing the entire 75 μL rxn mix immediately after incubation at 30 °C.
  7. 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.
  8. 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).

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