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.
E. coli-derived human BIM Long (BIM L) protein Ala2-Arg120, with an N-terminal 6-His tag
>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
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.
13-16 kDa and 30-35 kDa, reducing conditions
Read Publication using 1325-BL in the following applications:
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.
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.
Prepare dilutions of Recombinant Human BIML (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.
Aliquot 15 μL of each of the BIML 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 BIML (cleaved BID/Bcl-w control).
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.
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 BIML or Bcl‑w Minus C-Terminus (cleaved BID control).
Add 12 μL of mitochondria (approximately 25-30 μg) and 23 μL of Mitochondria Buffer with added protease inhibitors and BSA to each tube.
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.
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 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.
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
BIML (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. BIML 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. BIML 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 BIML from microtubules to neutralize anti-apoptotic Bcl-2 family members, allowing for the initiation of apoptosis.
O'Connor, L. et al. (1998) EMBO J. 17:384.
Puthalakath, H. et al. (1999) Mol. Cell. 3:287.
Miyashita, T. (2001) FEBS Letters 509:135.
Strasser, A. (2000) Ann N Y Acad Sci. 917:541.
Marani, M. et al. (2002) Mol. Cell. Biol. 22:3577.
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