Recombinant Human BIM (Long Form) Protein, CF

• Reactivity: Hu
• Applications: Bioactivity
• Format: Carrier-Free

Recombinant Human BIM (Long Form) Protein, CF Summary

• Details of Functionality: Measured by its ability to neutralize anti-apoptotic Bcl-2 family member inhibition of Recombinant Human BID Caspase-8-cleaved (Catalog # 882-B8)-mediated Cytochrome c release from isolated mitochondria. Release of Cytochrome c is quantified using the Rat/Mouse Cytochrome c Quantikine ELISA (Catalog # MCTC0). The typical EC₅₀ value in this assay is 0.1-2 µM.
  Optimal dose should be determined for each application.
• Source: E. coli-derived human BIM Long (BIM L) protein
  Ala2-Arg120, with an N-terminal 6-His tag
• Accession #: NP_006529
• N-terminal Sequence: His
• Protein/Peptide Type: Recombinant Proteins
• Gene: BCL2L11
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

Applications/Dilutions

• Dilutions:
  • Bioactivity
• Theoretical MW: 14.3 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: 13-16 kDa and 30-35 kDa, 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-KOH, DTT, KCl and Glycerol.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
• Recombinant Human BIM_L (Catalog # 1325-BL)
• Recombinant Human BID Caspase-8-cleaved (Catalog # 882-B8)
• Recombinant Human Bcl‑w Minus C-Terminus (Catalog # 824-BW)
• 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)
• Formulation Buffer: 20 mM Tris (pH 8.5), 150 mM NaCl, 1 mM Dtt, 20% Glycerol, 1 mg/mL fatty acid free BSA* (Sigma, Catalog # A6003)
• 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, (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 stock solution should be prepared at 100 mg/mL.
Note: All buffers, proteins and tubes should be kept on ice until indicated. Assay volumes are 75 μL and are combined in 0.5 mL Eppendorf tubes.
1. Prepare a stock solution of Recombinant Human Bcl‑w Minus C-Terminus (MW: 19.6 kDa) in Formulation Buffer at 59 μg/mL. The final concentration will be 600 nM.
2. Prepare dilutions of Recombinant Human BIM_L (MW: 14.3 kDa) in Formulation Buffer at concentrations of 15,000, 5000, 1500, 500, 150, 50, and 15 nM. The final concentration range will be 3000 to 3 nM in a total rxn volume of 75 μL.
3. Aliquot 15 μL of each of the BIM_L dilutions to a series of tubes. Add 15 μL of the Recombinant Human Bcl‑w Minus C-Terminus to each tube and gently mix. Incubate for 60 minutes at room temperature. Include one sample without Recombinant Human BIM_L (cleaved BID/Bcl-w control).
4. Prepare a stock solution of Recombinant Human BID Caspase-8-cleaved (MW: 22.0 kDa) in BID Dilution Buffer at 9.0 μg/mL. The final concentration will be 54 nM.
5. Add 10 μL of the Recombinant Human BID Caspase-8-cleaved Recombinant Human BID to each tube and gently mix. Incubate for 60 minutes at room temperature. Include one sample without Recombinant Human BIM_L or Bcl‑w Minus C-Terminus (cleaved BID control).
6. Add 12 μL of mitochondria (approximately 25-30 μg) and 23 μL of Mitochondria Buffer with added protease inhibitors and BSA to each tube.
7. 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.
8. Cap the tubes and gently mix the contents for 5-10 seconds. Incubate in a 30 °C water bath for 30 minutes.
9. Total Cytochrome c in the assay should be determined by freezing the entire 75 μL rxn mix immediately after incubation at 30 °C.
10. 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.
11. 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 BIM (Long Form) Protein, CF

• BAM
• BCL2L11
• BIM Long
• BIM
• BOD

Background

BIM_L (also known as Bod) is one of several splice variants of BIM, a pro-apoptotic protein belonging to the BH3 domain-only subgroup of Bcl-2 family members. BIM_L is thought to promote apoptosis by binding and inhibiting the activity of anti-apoptoticBcl-2 family members, thereby inducing the release of cytochrome c from mitochondria. BIM_L is normally sequestered in an inactive conformation from anti-apoptotic Bcl-2 family members through binding to the microtubule-associated dynein motor complex.Certain apoptotic stimuli release BIM_L from microtubules to neutralize anti-apoptotic Bcl-2 family members, allowing for the initiation of apoptosis.

1. O'Connor, L. et al. (1998) EMBO J. 17:384.
2. Puthalakath, H. et al. (1999) Mol. Cell. 3:287.
3. Miyashita, T. (2001) FEBS Letters 509:135.
4. Strasser, A. (2000) Ann N Y Acad Sci. 917:541.
5. Marani, M. et al. (2002) Mol. Cell. Biol. 22:3577.

Publications for BIM Long (1325-BL)(1)

Publication using 1325-BL

• Iwata A, de Claro RA, Morgan-Stevenson VL Extracellular Administration of BCL2 Protein Reduces Apoptosis and Improves Survival in a Murine Model of Sepsis. PLoS ONE, 2011-02-24;6(2):e14729. 2011-02-24 [PMID: 21390214] (In Vivo, Mouse)

Bioinformatics

• Gene Symbol: BCL2L11
• Uniprot:
  • NP_006529()