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Recombinant Human XIAP (BIR2) Protein, CF

Catalog #: 786-XB Datasheet
Catalog # Availability Size / Price Qty
786-XB-050
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Recombinant Human XIAP (BIR2) Protein, CF Summary

Product Specifications

Purity
>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
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 Recombinant Human (rh) Caspase-7 (Catalog # 823-C7) activity is 2-20 nM, as measured under the described conditions.
Source
E. coli-derived human XIAP protein
Ala124-Ala242, with an N-terminal Met and 6-His tag
Accession #
U45880
N-terminal Sequence
Analysis
Met
Predicted Molecular Mass
15 kDa
SDS-PAGE
12 kDa, reducing conditions

Product Datasheets

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786-XB

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.

786-XB

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% CHAPS, 10% Sucrose, pH 7.5
  • Assay Buffer: 25 mM HEPES, 0.1 M NaCl, 1 mM DTT, pH 8.0
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Recombinant Human Caspase‑7 (rhCaspase‑7) (Catalog # 823-C7)
  • Recombinant Human XIAP BIR2 Domain (rhXIAP-BIR2) (Catalog # 786-XB)
  • Substrate: Ac-Asp-Glu-Val-Asp-AFC (MP Biomedicals, Catalog # AFC138), 10 mM stock in DMSO
  1. Dilute rhXIAP-BIR2 (MW: 14,801Da) to various concentrations in Assay Buffer. Make serial dilutions of: 8000, 2000, 1000, 300, 100, 50, 30, 10, 3, and 1 nM.
  2. Dilute rhCaspase-7 to 0.12 ng/µL in Activation Buffer.
  3. Load Plate in the following order:
    70 µL Assay Buffer
    10 µL of the rhXIAP-BIR2 dilutions
    10 µL of 0.12 ng/µL rhCaspase-7
  4. Include a control, in duplicate, containing Assay Buffer and rhCaspase-7 only.
  5. Incubate at RT for 15 minutes.
  6. Dilute Substrate in Assay Buffer to 500 µM.
  7. Load 10 µL Substrate per well to start the 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) for rhXIAP-BIR2 by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.

  10. The specific activity for rhCaspase-7 at each point may be determined using the following formula (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: 0.0012 μg
  • rhXIAP-BIR2 curve: 800, 200, 100, 30, 10, 5, 3, 1, 0.3 and 0.1 nM
  • Substrate: 50 µM

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 ability of XIAP to inhibit caspases is prevented by SMAC/Diablo through binding to XIAP-BIR2 and -BIR3 domains.

 

SUPPLEMENTARY METHODS 

 

XIAP (BIR2 domain) inhibits DEVD-AFC cleavage by activated cell extracts.

 

XIAP (BIR2 domain) 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 32 pg/uL in Step 2.

The IC50 for inhibition of rhCaspase-3 activity is typically 10-100 nM.

 

References
  1. Deveraux, Q. et al. (1998) EMBO J. 17(8):2215.
  2. Deveraux, Q. et al. (1999) EMBO J. 18(19):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(29):27058.
  7. Ekert, P. et al. (2001) J. Cell Biol. 152(3):483.
  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

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