Recombinant Human Bcl-w (minus C-Terminus) Protein, CF

• Reactivity: Hu
• Applications: Inhibition Activity
• Format: Carrier-Free

Recombinant Human Bcl-w (minus C-Terminus) Protein, CF Summary

• Details of Functionality: 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 is between 50 and 250 nM in the presence of 54 nM Recombinant Human BID Caspase-8-cleaved (Catalog # 882-B8).
  Optimal dilutions should be determined by each laboratory for each application.
• Source: E. coli-derived human Bcl-w protein
  Ala2-Thr172, with a C-terminal 6-His tag
• Accession #: Q92843
• N-terminal Sequence: Ala2
• Structure / Form: Monomer and disulfide-linked homodimer
• Protein/Peptide Type: Recombinant Proteins
• Gene: BCL2L2
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Endotoxin Note: <0.01 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Inhibition Activity
• Theoretical MW: 19.5 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 18 kDa, under reducing and non-reducing conditions

Packaging, Storage & Formulations

• 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.
• Buffer: Supplied as a 0.2 μm filtered solution in HEPES, NaCl and Glycerol.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• 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.
  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 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.
  3. 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).
  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).

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Bcl-w (minus C-Terminus) Protein, CF

• Apoptosis regulator Bcl-W
• BCL2L2
• Bcl2-L-2
• BCL2-like 2
• Bclw
• Bcl-w
• BCLWbcl-2-like protein 2
• KIAA0271BCL2-L-2

Background

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.

1. Gross, A. et al. (1999) Genes and Develop. 13:1899.
2. Kroemer, G. (1997) Nature Med. 3:614.
3. Ross, J.A., et al. (1998) Nat. Genet. 18:251.
4. Print, C.G. et al. (1998) Proc. Natl. Acad. Sci. USA 95:12323.
5. Yan, W. et al. (2000) Mol. Endocrin. 14:682.

Publications for BCL-W/BCL2L2 (824-BW)(1)

Publication using 824-BW

• Cosker , Katharin, Pazyra-Murphy , Maria F, Fenstermacher , Sara J, Segal , Rosalind Target-derived neurotrophins coordinate transcription and transport of bclw to prevent axonal degeneration. J Neurosci, 2013-03-20;33(12):5195-207. 2013-03-20 [PMID: 23516285] (Bioassay, Mouse)

Bioinformatics

• Gene Symbol: BCL2L2
• Uniprot:
  • Q92843()