Recombinant Human XIAP (Full Length) Protein, CF

R&D Systems | Catalog # 822-XF

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Key Product Details

  • R&D Systems E. coli-derived Recombinant Human XIAP (Full Length) Protein (822-XF)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

E. coli

Accession Number

AAC50373

Applications

Inhibition Activity
View all XIAP Proteins and Enzymes »

Product Specifications

Source

E. coli-derived human XIAP protein
ATVID 6-His tag SSNG Human XIAP
(Met1-Ser497)
Accession # AAC50373
N-terminus C-terminus

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

Ala

Predicted Molecular Mass

58 kDa

SDS-PAGE

50-60 kDa, reducing conditions

Activity

Measured by its ability to inhibit DEVD-AFC cleavage activity by Recombinant Human Caspase‑7 (Catalog # 823-C7).
The IC50 for inhibition of rhCaspase-7 activity is <50 nM, as measured under the described conditions. 

Formulation, Preparation, and Storage

822-XF
Formulation Supplied as a 0.2 μm filtered solution in HEPES, NaCl, DTT 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.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.

Background: XIAP

XIAP (X-chromosome linked inhibitor of apoptosis) is a member of the inhibitor of apoptosis (IAP) family of proteins that inhibit caspases. The BIR2 domain of XIAP inhibits Caspase-3 and Caspase-7. The BIR3 domain inhibits Caspase-9. The ability of XIAP to inhibit caspases is prevented by SMAC/Diablo through binding to XIAP‑BIR2 and BIR3 domains.

 

SUPPLEMENTARY METHODS

XIAP inhibits DEVD-AFC cleavage by activated cell extracts.

See http://www.RnDSystems.com/go/IAPProtocols">www.RnDSystems.com/go/IAPProtocols

 

XIAP inhibits DEVD-AFC cleavage by Recombinant Human Caspase-3.

Recombinant Human Caspase‑3 (Catalog # http://www.rndsystems.com/product_results.aspx?k=707-C3">707-C3) can be used in the Activity Assay Protocol in place of Casapase-7 by diluting to initial concentration of 0.1 ng/μL in Step 1. 

The IC50 for inhibition of rhCaspase-3 activity is typically 10-200 nM.

References

  1. Deveraux, Q. et al. (1998) EMBO J. 17:2215.
  2. Deveraux, Q. et al. (1999) EMBO J. 18:5242.
  3. Verhagen, A. et al. (2000) Cell 102:43.
  4. Chai, J. et al. (2001) Cell 104:769.
  5. Riedl, S. (2001) Cell 104:791.
  6. Suzuki, Y. (2001) J. Biol. Chem. 276:27058.
  7. Ekert, P. et al. (2001) J. Cell Biol. 152:48.
  8. Du, C. et al. (2000) Cell 102:33.

Long Name

X-linked Inhibitor of Apoptosis

Alternate Names

BIRC4

Entrez Gene IDs

331 (Human); 11798 (Mouse); 63879 (Rat)

Gene Symbol

XIAP

UniProt

AAC50373

Additional XIAP Products

Product Documents for Recombinant Human XIAP (Full Length) Protein, CF

Certificate of Analysis

To download a Certificate of Analysis, please enter a lot or batch number in the search box below.

Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Recombinant Human XIAP (Full Length) Protein, CF

For research use only

Related Research Areas

Cysteine Proteases and Regulators

Citations for Recombinant Human XIAP (Full Length) Protein, CF

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Protocols

View specific protocols for Recombinant Human XIAP (Full Length) Protein, CF (822-XF):

Materials
  • Activation Buffer: 50 mM HEPES, 100 mM NaCl, 1 mM EDTA, 5 mM DTT, 0.1% (w/v) CHAPS, 10% Sucrose, pH 7.5
  • Assay Buffer: 25 mM HEPES, 100 mM NaCl, pH 8.0
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Recombinant Human Caspase‑7 (rhCaspase-7) (Catalog # 823-C7)
  • Recombinant Human XIAP Full Length (rhXIAP) (Catalog # 822-XF)
  • Substrate:  Ac-Asp-Glu-Val-Asp-AFC (MP Biomedicals, Catalog # AFC138), 10 mM stock in DMSO
  1. Dilute rhCaspase-7 to 0.2 ng/µL in Activation Buffer.
  2. Dilute rhXIAP (MW: 58,000 Da) to various concentrations in Assay Buffer. Make serial dilutions of: 12000, 5000, 1500, 500, 150, 50, 15, 5 and 1 nM.
  3. Load Plate with the following:
       10 µL of the rhXIAP dilution curve
       10 µL of 0.2 ng/µL rhCaspase-7
       70 µL Assay Buffer
  4. Include a control, in duplicate containing Assay Buffer and rhCaspase-7 only.
  5. Incubate plate at RT for 15 minutes.
  6. Dilute Substrate to 500 µM in DMSO.
  7. Load 10 µL Substrate per well to start reaction.
  8. Read at excitation and emission wavelengths of 400 nm and 505 nm, respectively in kinetic mode for 5 minutes.
  9. Derive the 50% inhibiting concentration (IC50) of rhXIAP by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
  10. The specific activity of rhCaspase-7 at each point may be determined using the following equation (if needed):

     Specific Activity (pmol/min/µg) =

 Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)
amount of enzyme (µg)

     *Adjusted for Substrate Blank
     **Derived using calibration standard 7-amino, 4-trifluoromethylcoumarin (AFC) (Calbiochem, Catalog # 164580)

Per Well:

  • rhCaspase-7: 0.002 µg
  • rhXIAP: 1200, 500, 150, 50, 15, 5, 1.5, 0.5, and 0.1 nM
  • Substrate: 50 µM

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