Recombinant Human XIAP (Full Length) Protein, CF Summary
|ATVID||6-His tag||SSNG||Human XIAP |
Accession # AAC50373
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, 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.
- 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
- Dilute rhCaspase-7 to 0.2 ng/µL in Activation Buffer.
- 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.
- 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
- Include a control, in duplicate containing Assay Buffer and rhCaspase-7 only.
- Incubate plate at RT for 15 minutes.
- Dilute Substrate to 500 µM in DMSO.
- Load 10 µL Substrate per well to start reaction.
- 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 by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
- 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)
- rhCaspase-7: 0.002 µg
- rhXIAP: 1200, 500, 150, 50, 15, 5, 1.5, 0.5, and 0.1 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 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.
XIAP inhibits DEVD-AFC cleavage by activated cell extracts.
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.
- Deveraux, Q. et al. (1998) EMBO J. 17:2215.
- Deveraux, Q. et al. (1999) EMBO J. 18:5242.
- 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. 276:27058.
- Ekert, P. et al. (2001) J. Cell Biol. 152:48.
- Du, C. et al. (2000) Cell 102:33.
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.
Citations: Showing 1 - 3
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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., 2020-05-06;0(0):.
Applications: Cell Culture
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, 2013-12-26;28(4):1938-46.
Sample Types: Recombinant Protein
Applications: Binding Assay, Ubiquitination
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., 2007-07-31;361(4):994-9.
Applications: Surface Plasmon Resonance
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