Recombinant Human XIAP (Full Length) Protein, CF

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822-XF-050
R&D Systems Recombinant Proteins and Enzymes
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Recombinant Human XIAP (Full Length) Protein, CF Summary

Product Specifications

Purity
>85%, by SDS-PAGE under reducing conditions and visualized by silver stain.
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
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. 
Source
E. coli-derived human XIAP protein
ATVID 6-His tag SSNG Human XIAP
(Met1-Ser497)
Accession # AAC50373
N-terminus C-terminus
Accession #
N-terminal Sequence
Analysis
Ala
Predicted Molecular Mass
58 kDa
SDS-PAGE
50-60 kDa, reducing conditions

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Product Datasheets

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822-XF

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.

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.

Assay Procedure

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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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 www.RnDSystems.com/go/IAPProtocols

 

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

Recombinant Human Caspase‑3 (Catalog # 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
Entrez Gene IDs
331 (Human); 11798 (Mouse); 63879 (Rat)
Alternate Names
API3X-linked inhibitor of apoptosis protein; apoptosis inhibitor 3; baculoviral IAP repeat-containing 4; baculoviral IAP repeat-containing protein 4; BIRC4; BIRC4XLP2; E3 ubiquitin-protein ligase XIAP; EC 6.3.2.-; hIAP3; hIAP-3; hILP; IAP3; IAP-3; IAP-like protein; ILP; ILP1; Inhibitor of apoptosis protein 3; MIHA; XIAP; X-linked IAP; X-linked inhibitor of apoptosis

Citations for Recombinant Human XIAP (Full Length) 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.

3 Citations: Showing 1 - 3
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  1. Monoglyceride lipase mediates tumor-suppressive effects by promoting degradation of X-linked inhibitor of apoptosis protein
    Authors: R Liu, X Wang, C Curtiss, MS Sheikh, Y Huang
    Cell Death Differ., 2020;0(0):.
    Species: Human
    Sample Types:
    Applications: Cell Culture
  2. A novel role for inhibitor of apoptosis (IAP) proteins as regulators of endothelial barrier function by mediating RhoA activation.
    Authors: Hornburger M, Mayer B, Leonhardt S, Willer E, Zahler S, Beyerle A, Rajalingam K, Vollmar A, Furst R
    FASEB J, 2013;28(4):1938-46.
    Species: Human
    Sample Types: Recombinant Protein
    Applications: Binding Assay
  3. 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;361(4):994-9.
    Species: Human
    Sample Types:
    Applications: Surface Plasmon Resonance

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