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Recombinant Human Bcl-2 related protein A1 (aa 1-152), CF

Catalog #: 1160-A1
2 Citations Datasheet / COA / SDS
Catalog # Availability Size / Price Qty
1160-A1-050
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Recombinant Human Bcl-2 related protein A1 (aa 1-152), CF Summary

Product Specifications

Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
Activity
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 human  Bcl‑2 related protein A1 Minus C-terminus in this assay is 15-200 nM.
Source
E. coli-derived human Bcl-2 related protein A1 protein
Thr2-Ser152, with a C-terminal 6-His tag
Accession #
Q16548
N-terminal Sequence
Analysis
Thr2
Structure / Form
Monomer
Predicted Molecular Mass
18 kDa
SDS-PAGE
16 kDa, non-reducing conditions

Product Datasheets

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1160-A1

Product Datasheet
COA
SDS

Carrier Free

What does CF mean?

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.

What formulation is right for me?

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.

1160-A1

Formulation Supplied as a 0.2 μm filtered solution in NaH2PO4, 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.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.

Assay Procedure

Materials
  • Recombinant Human Bcl‑2 related protein A1 Minus C-terminus (Recombinant Human BCL2A1) (Catalog # 1160-A1)
  • Recombinant Human BID Caspase-8-cleaved (Catalog # 882-B8)
  • 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, 10% Glycerol, 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 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.
  2. Prepare dilutions of Recombinant Human BCL2A1 (MW: 18.3 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 rxn volume of 75 μL.
  3. Aliquot 25 μL of each of the BCL2A1 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 BCL2A1 (cleaved BID control).
  4. Add 12 μL of mitochondria (approximately 25-30 μg) and 28 μL of Mitochondria Buffer containing protease inhibitors and BSA to each tube.
  5. 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.
  6. Cap the tubes and gently mix the contents for 5-10 seconds. Incubate in a 30 °C water bath for 30 min.
  7. Total Cytochrome c in the assay should be determined by freezing the entire 75 μL rxn mix immediately after incubation at 30 °C.
  8. 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.
  9. 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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Background: Bcl-2 related protein A1

Bcl‑2 related protein A1 (BCL2A1) is a member of the Bcl-2 family of proteins that regulate the outer mitochondrial membrane permeability. BCL2A1 is an anti‑apoptotic member that prevents release of cytochrome c from the mitochondria intermembrane space into the cytosol. BCL2A1 is present on the outer mitochondrial membrane and is also found on other intracellular membranes in some cell types. Natural BCL2A1 contains a carboxyl-terminal mitochondria targeting sequence. Recombinant BCL2A1 is missing the mitochondrial targeting sequence but maintains its ability to neutralize pro-apoptotic Bcl-2 family members. Neutralization by BCL2A1 appears to occur by binding to the BH3 region of pro-apoptotic Bcl-2 family members. This activity does not require the mitochondrial targeting sequence.

References
  1. Duriez, P. et al. (2000) J. Biol. Chem. 275(24):18099.
  2. Holmgreen, S. et al. (1999) Cell Death and Diff. 6:525.
  3. Lin, Ey (1993) J. Immunol. 151:197.
  4. Karsan, A. (1996) Blood 87:3089.
Entrez Gene IDs
597 (Human); 12044 (Mouse)
Alternate Names
ACC-1; ACC-2; Bcl-2 related protein A1; BCL2A1; Bcl2L5; Bcl2-L-5; BCL2L5HBPA1; Bcl-2-like protein 5; BCL2-related protein A1; Bfl-1; BFL1bcl-2-related protein A1; GRS; GRSbcl2-L-5; hematopoietic BCL2-related protein A1; Hemopoietic-specific early response protein; Protein BFL-1; Protein GRS

Citations for Recombinant Human Bcl-2 related protein A1 (aa 1-152), 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.

2 Citations: Showing 1 - 2
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  1. Modulation of Bax and mTOR for cancer therapeutics
    Authors: R Li, C Ding, J Zhang, M Xie, D Park, Y Ding, G Chen, G Zhang, M Gilbert-Ro, W Zhou, A Marcus, SY Sun, ZG Chen, GL Sica, SS Ramalingam, A Magis, H Fu, F Khuri, WJ Curran, TK Owonikoko, DM Shin, J Zhou, X Deng
    Cancer Res., 2017-04-05;0(0):.
    Species: Human
    Sample Types: Whole Cells
    Applications: Bioassay
  2. Extracellular Administration of BCL2 Protein Reduces Apoptosis and Improves Survival in a Murine Model of Sepsis.
    Authors: Iwata A, de Claro RA, Morgan-Stevenson VL
    PLoS ONE, 2011-02-24;6(2):e14729.
    Species: Mouse
    Sample Types: In Vivo
    Applications: In Vivo

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