Measured by its ability to reverse the inhibition of DEVD-AFC cleavage activity in cell extracts activated by addition of cytochrome c and dATP. The IC50 for reversal of XIAP-BIR3 (100 nM) inhibition of DEVD-AFC cleavage in activated cell extracts is <2,000 nM. Optimal dilutions should be determined by each laboratory for each application.
E. coli-derived Ala56-Asp239, with a C-terminal 6-His tag
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
22 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.
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
Note: All reagents and assay components should be kept on ice until use.
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:
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Human SMAC/Diablo Protein, CF
diablo, IAP-binding mitochondrial protein
Direct IAP-binding protein with low pI
mitochondrial Smac protein
Second mitochondria-derived activator of caspase
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.
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.
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