Recombinant Human XIAP (BIR3) Protein, CF Summary
Optimal dilutions should be determined by each laboratory for each application.
Asn252-Thr356, with an N-terminal Met and 6-His tag
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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.
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.
|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.
- 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
- 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.
- 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.
- 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.
- 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:
- Total Control: 10 μL of Extraction Buffer, 10 μL of cell extract supernatant, and 5 μL of the cytochrome C/dATP activator mixture.
- Inactive Control: 15 μL of Extraction Buffer and 10 μL of cell extract supernatant. The total reaction volume is 25 μL.
- Incubate for 60 minutes at 37 °C.
- After incubation, add 100 μL of Assay Buffer to each vial for a five-fold dilution. Mix briefly.
- Dilute Substrate to 100 μM in Assay Buffer.
- In a plate load 50 μL of diluted incubated reaction mixtures, and start the reaction by adding 50 μL of 100 μM Substrate.
- Read at excitation and emission wavelengths of 400 nm and 505 nm, respectively, in kinetic mode for 5 minutes.
- 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.
- Normalized activity may be determined using the following equation:
% Normalized Activity = Sample (RFU/min) - Inactive Control** (RFU/min) x 100% Total Control (RFU/min)
- rhXIAP (BIR3) curve: 2000, 1000, 600, 200, 100, 20, 10, and 2 nM
- Substrate: 50 μM
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.
- Deveraux, Q. et al. (1998) EMBO J. 17(8):2215.
- Deveraux, Q. et al. (1999) EMBO J. 18(19):5242.
- Verhagen, A. et al. (2000) Cell 102:43.
- Chai, J. et al. (2001) Cell 104:769.
- Riedl, S. (2001) Cell 104:791.
- Suzuki, Y. (2001) J. Biol. Chem. 276(29):27058.
- Ekert, P. et al. (2001) J. Cell Biol. 152(3):483.
- Du, C. et al. (2000) Cell 102:33.
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.
Citations: Showing 1 - 2
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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;8(1):13549.
Sample Types: Cell Lysates
Applications: Western Blot
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;108(8):1657-1666.
Sample Types: Whole Cells
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