Recombinant Human SMAC/Diablo Protein, CF Summary
Product Specifications
Optimal dilutions should be determined by each laboratory for each application.
Ala56-Asp239, with a C-terminal 6-His tag
Analysis
Product Datasheets
Carrier Free
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.
789-SM
| Formulation | Supplied as a 0.2 μm filtered solution in HEPES and NaCl. |
| 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.
|
Assay Procedure
- Jurkat E6 wild type cell extracts (see supplementary methods for preparation)
- Extraction Buffer: 50 mM HEPES, 10 mM KCl, 5 mM EGTA, 1 mM MgCl2, 0.2% CHAPS, 0.2 mM DTT, pH 7.5
- Assay Buffer: 10 mM HEPES, 0.5 mM EGTA, 5 mM DTT, 10% Glycerol, pH 7.5
- Formulation Buffer: 25 mM HEPES, 0.1 M KCl, pH 7.5
- Recombinant Human SMAC/Diablo (rhSMAC) (Catalog # 789-SM)
- Recombinant Human XIAP BIR3 Domain (rhXIAP) (Catalog # 895-XB)
- Cytochrome C, Bovine heart (Sigma, Catalog # C3131), 2 mg/mL stock in deionized water
- dATP (Sigma, Catalog # D6500), 10 mM stock adjusted to pH 7.5 with NaOH
- Substrate: Ac-Asp-Glu-Val-Asp-AFC (DEVD-AFC) (MP Biomedicals, Catalog # AFC138), 10 mM stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Thaw cell extracts and centrifuge in a microcentrifuge at 14,000 rpms for 5 minutes at 4 °C. Transfer supernatants to chilled tubes and use within 1 hour.
- Prepare a 250 nM stock of rhXIAP (BIR3) (MW: 13.0 KDa) in Formulation Buffer.
- Prepare a curve of rhSMAC (MW: 21.6 KDa ) in Formulation Buffer. Make the following serial dilutions: 50,000, 15,000, 7500, 3000, 1500, 600 and 300 nM. Note: High point may not be achievable depending on lot received.
- Prepare the activator mixture by combining equal volumes of 2 mg/mL Cytochrome C and 10 mM dATP for working concentrations of 1 mg/mL and 5 mM, respectively.
- Prepare reaction mixtures in tubes by combining 5 μL of each rhSMAC curve dilution, 5 μL of rhXIAP (BIR3), 10 μL of cell extract supernatant, and 5 μL of the cytochrome C/dATP activator mixture. Also, prepare the following controls:
- Total Control: 10 μL of Extraction Buffer, 10 μL of cell extract supernatant, and 5 μL of the cytochrome C/dATP activator mixture.
- Inactive Control: 15 μL of Extraction Buffer and 10 μL of cell extract supernatant. The total reaction volume is 25 μL.
- rhXIAP (BIR3) only Conrol: 5 μL of Extraction Buffer, 5 μL of rhXIAP (BIR3), 10 μL of cell extract supernatant, and 5 μL of the cytochrome C/dATP activator mixture.
- Incubate for 60 minutes at 30 °C.
- After incubation, add 100 μL of Assay Buffer to each vial for a five-fold dilution. Mix briefly.
- Dilute Substrate to 100 μM in Assay Buffer.
- In a plate load 50 μL of diluted incubated reaction mixtures and start the reaction by adding 50 μL of 100 μM Substrate.
- Read at excitation and emission wavelengths of 400 nm and 505 nm, respectively, in kinetic mode for 5 minutes.
- Derive the 50% inhibiting concentration (IC50) of rhSMAC by plotting normalized activity vs. reaction concentration of rhSMAC with 4‑PL fitting.
- Normalized activity may be determined using the following equation:
|
% Normalized Activity = |
Sample (RFU/min) - Inactive Control (RFU/min) |
x 100% |
|
Total Control (RFU/min) |
- rhSMAC curve: 10,000, 3000, 1500, 600, 300, 120 and 60 nM
Reconstitution Calculator
Background: SMAC/Diablo
SMAC (second mitochondria derived activator of caspase)/Diablo promotes caspase activation by interacting with the inhibitor of apoptosis (IAP) proteins in the cytochrome c/Apaf-1/caspase-9 pathway.
SUPPLEMENTARY METHODS
SMAC/Diablo can reverse rhXIAP inhibition of DEVD-AFC cleavage in activated cell lystes.
Recombinant Human XIAP Full Length (Catalog # 822-XF) can be used in the Activity Assay Protocol in place of rhXIAP (BIR3). The IC50 for reversal of XIAP (500 nM) inhibition of DEVD-AFC cleavage in activated cell extracts is typically 500-1500 nM.
- Du, C. et al. (2000) Cell 102:33.
- Chai, J. et al. (2000) Nature 406:855.
- Srinivasula, S. et al. (2000) J. Biol. Chem. 275:36152.
- Ekert, P. et al. (2001) J. Cell Biol. 152:483.
- Verhagen, A. (2000) Cell 102:43.
Citations for Recombinant Human SMAC/Diablo 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.
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Citations: Showing 1 - 2
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Phosphorylation of Smac by JNK3 attenuates its interaction with XIAP.
Authors: Park BD, Ham YM, Jeong HJ, Cho SJ, Je YT, Yoo KD, Lee SK
Biochem. Biophys. Res. Commun., 2007-07-31;361(4):994-9.
Species: Human
Sample Types:
Applications: Surface Plasmon Resonance -
Survivin interacts with Smac/DIABLO in ovarian carcinoma cells but is redundant in Smac-mediated apoptosis.
Authors: McNeish IA, Lopes R, Bell SJ, McKay TR, Fernandez M, Lockley M, Wheatley SP, Lemoine NR
Exp. Cell Res., 2005-01-01;302(1):69-82.
Species: Human
Sample Types:
Applications: Bioassay
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