Recombinant Human SMAC/Diablo Protein, CF

• Reactivity: Hu
• Applications: Inhibition Activity
• Format: Carrier-Free

Recombinant Human SMAC/Diablo Protein, CF Summary

• Details of Functionality: 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 (50 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.
• Source: E. coli-derived human SMAC/Diablo protein
  Ala56-Asp239, with a C-terminal 6-His tag
• Accession #: Q9NR28
• N-terminal Sequence: Ala56
• Protein/Peptide Type: Recombinant Enzymes
• Gene: DIABLO
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.

Applications/Dilutions

• Dilutions:
  • Inhibition Activity
• Theoretical MW: 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.
• SDS-PAGE: 23 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 and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• 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
  Note: All reagents and assay components should be kept on ice until use.
  1. 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.
  2. Prepare a 250 nM stock of rhXIAP (BIR3) (MW: 13.0 KDa) in Formulation Buffer.
  3. 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.
  4. 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.
  5. 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:
     
     1. Total Control: 10 μL of Extraction Buffer, 10 μL of cell extract supernatant, and 5 μL of the cytochrome C/dATP activator mixture.
     2. Inactive Control: 15 μL of Extraction Buffer and 10 μL of cell extract supernatant. The total reaction volume is 25 μL.
     3. 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.
  6. Incubate for 60 minutes at 30 °C.
  7. After incubation, add 100 μL of Assay Buffer to each vial for a five-fold dilution. Mix briefly.
  8. Dilute Substrate to 100 μM in Assay Buffer.
  9. In a plate load 50 μL of diluted incubated reaction mixtures and start the reaction by adding 50 μL of 100 μM Substrate.
  10. Read at excitation and emission wavelengths of 400 nm and 505 nm, respectively, in kinetic mode for 5 minutes.
  11. Derive the 50% inhibiting concentration (IC₅₀) of rhSMAC by plotting normalized activity vs. reaction concentration of rhSMAC with 4‑PL fitting.
  12. Normalized activity may be determined using the following equation:

  % Normalized Activity =	Sample (RFU/min) - Inactive Control (RFU/min)	x 100%
  	Total Control (RFU/min)

  Per Reaction:
  • rhSMAC curve: 10,000, 3000, 1500, 600, 300, 120 and 60 nM

Notes

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

• 0610041G12Rik
• Diablo
• diablo, IAP-binding mitochondrial protein
• Direct IAP-binding protein with low pI
• mitochondrial Smac protein
• Second mitochondria-derived activator of caspase
• SMAC
• SMACmitochondrial

Background

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.

1. Du, C. et al. (2000) Cell 102:33.
2. Chai, J. et al. (2000) Nature 406:855.
3. Srinivasula, S. et al. (2000) J. Biol. Chem. 275:36152.
4. Ekert, P. et al. (2001) J. Cell Biol. 152:483.
5. Verhagen, A. (2000) Cell 102:43.

Publications for SMAC/Diablo (789-SM)(2)

Publications using 789-SM

• Park BD, Ham YM, Jeong HJ, Cho SJ, Je YT, Yoo KD, Lee SK Phosphorylation of Smac by JNK3 attenuates its interaction with XIAP. Biochem. Biophys. Res. Commun., 2007-07-31;361(4):994-9. 2007-07-31 [PMID: 17686459] (Surface Plasmon Resonance, Human)
• McNeish IA, Lopes R, Bell SJ, McKay TR, Fernandez M, Lockley M, Wheatley SP, Lemoine NR Survivin interacts with Smac/DIABLO in ovarian carcinoma cells but is redundant in Smac-mediated apoptosis. Exp. Cell Res., 2005-01-01;302(1):69-82. 2005-01-01 [PMID: 15541727] (Bioassay, Human)

Bioinformatics

• Gene Symbol: DIABLO
• Uniprot:
  • Q9NR28()