Home / SMAC/Diablo / Recombinant Human SMAC/Diablo Protein, CF

Recombinant Human SMAC/Diablo Protein, CF

Catalog #: 789-SM
2 Citations Datasheet
Catalog # Availability Size / Price Qty
789-SM-100
R&D Systems Recombinant Proteins and Enzymes
1 Image
Product Details
Citations (2)
FAQs
Supplemental Products
Reviews
Summary Product Datasheets Carrier Free Data Images Reconstitution Calculator Background Related Research Areas

Recombinant Human SMAC/Diablo Protein, CF Summary

Product Specifications

Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
Activity
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
Analysis
Ala56
Predicted Molecular Mass
22 kDa
SDS-PAGE
23 kDa, reducing conditions

Product Datasheets

You must select a language.

x

789-SM

Product Datasheet

Carrier Free

What does CF mean?

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.

What formulation is right for me?

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.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.

Assay Procedure

Materials
  • 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

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.

References
  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.
Long Name
Second Mitochondria-derived Activator of Caspase
Entrez Gene IDs
56616 (Human)
Alternate Names
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

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.

2 Citations: Showing 1 - 2
Filter your results:

Filter by:

  1. 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
  2. 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

FAQs

No product specific FAQs exist for this product, however you may

View all Proteins and Enzyme FAQs
Loading...

Reviews for Recombinant Human SMAC/Diablo Protein, CF

There are currently no reviews for this product. Be the first to review Recombinant Human SMAC/Diablo Protein, CF and earn rewards!

Have you used Recombinant Human SMAC/Diablo Protein, CF?

Submit a review and receive an Amazon gift card.

$25/€18/£15/$25CAN/¥75 Yuan/¥2500 Yen for a review with an image

$10/€7/£6/$10 CAD/¥70 Yuan/¥1110 Yen for a review without an image

Submit a Review