Recombinant Human XIAP (BIR3) Protein, CF

Catalog # Availability Size / Price Qty
895-XB-050
R&D Systems Recombinant Proteins and Enzymes
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Recombinant Human XIAP (BIR3) Protein, CF Summary

Product Specifications

Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
Activity
Measured by its ability to inhibit DEVD-AFC cleavage activity in cell extracts activated by addition of cytochrome c and dATP. The IC50 for this effect is <300 nM.
Optimal dilutions should be determined by each laboratory for each application.
Source
E. coli-derived human XIAP protein
Asn252-Thr356, with an N-terminal Met and 6-His tag
Accession #
N-terminal Sequence
Analysis
Met
Predicted Molecular Mass
13 kDa
SDS-PAGE
11 kDa, reducing conditions

Product Datasheets

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895-XB

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.

895-XB

Formulation Supplied as a 0.2 μm filtered solution in HEPES, NaCl and DTT.
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 NaCl, 1 mM DTT, pH 8.5
  • Recombinant Human XIAP BIR3 Domain  (rhXIAP) (BIR3) (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 curve of rhXIAP (BIR3) MW: 13,047 Da) in Formulation Buffer. Make the following serial dilutions: 5000, 2500, 1500, 500, 250, 50, 25, and 5 nM. Note: High point may not be achievable depending on lot received.
  3. 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.
  4. Prepare reaction mixtures in tubes by combining 10 μL of each rhXIAP (BIR3)  curve dilution, 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.
  5. Incubate for 60 minutes at 37 °C.
  6. After incubation, add 100 μL of Assay Buffer to each vial for a five-fold dilution. Mix briefly.
  7. Dilute Substrate to 100 μM in Assay Buffer.
  8. In a plate load 50 μL of diluted incubated reaction mixtures, and start the reaction by adding 50 μL of 100 μM Substrate.
  9. Read at excitation and emission wavelengths of 400 nm and 505 nm, respectively, in kinetic mode for 5 minutes.
  10. Derive the 50% inhibiting concentration (IC50) of rhXIAP (BIR3)  by plotting normalized activity vs. reaction concentration of rhXIAP (BIR3) (step 4) with 4-PL fitting.
  11. 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:
  • rhXIAP (BIR3) curve:  2000, 1000, 600, 200, 100, 20, 10, and 2 nM
  • Substrate: 50 μM
Reconstitution Calculator

Reconstitution Calculator

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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 BIR3 domain inhibits caspase-9. The ability of XIAP (BIR3 domain) to inhibit caspases is prevented by SMAC/Diablo.

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

Citations for Recombinant Human XIAP (BIR3) 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
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  1. Pharmacological difference between degrader and inhibitor against oncogenic BCR-ABL kinase
    Authors: N Shibata, K Shimokawa, K Nagai, N Ohoka, T Hattori, N Miyamoto, O Ujikawa, T Sameshima, H Nara, N Cho, M Naito
    Sci Rep, 2018-09-10;8(1):13549.
    Species: Human
    Sample Types: Cell Lysates
    Applications: Western Blot
  2. Development of protein degradation inducers of oncogenic BCR-ABL protein by conjugation of ABL kinase inhibitors and IAP ligands
    Authors: N Shibata, N Miyamoto, K Nagai, K Shimokawa, T Sameshima, N Ohoka, T Hattori, Y Imaeda, H Nara, N Cho, M Naito
    Cancer Sci., 2017-06-19;108(8):1657-1666.
    Species: Human
    Sample Types: Whole Cells
    Applications: Bioassay

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