Recombinant Human BIM (Long Form) Protein, CF Summary
Optimal dose should be determined for each application.
Ala2-Arg120, with an N-terminal 6-His tag
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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.
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.
|Formulation||Supplied as a 0.2 μm filtered solution in HEPES-KOH, DTT, KCl 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.
*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.
- 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.
- 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).
Background: BIM Long (BIM L)
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.
Citation for Recombinant Human BIM (Long Form) Protein, 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.
1 Citation: Showing 1 - 1
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;6(2):e14729.
Sample Types: In Vivo
Applications: In Vivo
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Quantikine Colorimetric Sandwich ELISAs
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