Recombinant Human cIAP-1 (HIAP-2) Protein, CF Summary
Product Specifications
Optimal dilutions should be determined by each laboratory for each application.
| ATVID | 10-His tag | SIEGRA | Human cIAP-1 (His2-Ser618) Accession # Q13490 |
| N-terminus | C-terminus | ||
Analysis
Product Datasheets
818-IA
Carrier Free
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.
818-IA
| 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.
|
Assay Procedure
- Jurkat A3 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
- Recombinant Human cIAP‑1/HIAP‑2 (rhcIAP-1) (Catalog # 818-IA)
- 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
- Cell Extracts from Jurkat A3 wild type cells (see above protocol)
- Substrate: Ac-Asp-Glu-Val-Asp-AFC (DEVD-AFC) (MP Biomendicals, 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 rhcIAP-1 (MW: 72,000 Da) in Extraction Buffer. Make the following serial dilutions: 5000, 2000, 1000, 500, 250, 125, 62.5, and 12.5 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 rhcIAP-1 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 30 °C.
- After incubation, add 100 μL of Assay Buffer to each vial for a 1/5 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 rhcIAP-1 by plotting normalized activity vs. reaction concentration of rhcIAP-1 (step 4) with Semi-Log Fitting [y = A + B * Log(x)]. Solve for x when y = 50.
- Normalized activity may be determined using the following equation:
| % Normalized Activity = | Sample (RFU/min) - Inactive Control** (RFU/min) | x 100% |
| Total Control (RFU/min) |
- rhcIAP-1 curve: 2000, 800, 400, 200, 100, 50, 25, and 5 nM
- Substrate: 50 μM
Background: cIAP-1/HIAP-2
cIAP-1 (also known as MIHB and HIAP-2) is a member of the inhibitor of apoptosis (IAP) family of proteins that inhibit the proteolytic activity of mature caspases. cIAP-1 has 3 BIR (baculovirus inhibitor of apoptosis) domains, a RING finger domain, and a caspase recruitment domain (CARD). cIAP-1 inhibits caspases by interaction of the BIR domain with the active caspase. Caspase activity may be restored through interactions with the Reaper like motif on mitochondrial proteins such as SMAC/Diablo or HTRA-2/Omi. cIAP-1 is reported to be cleaved by caspases in fetal rat hepatocytes treated with TGF-beta.
- Roy, N. et al. (1997) EMBO J. 23:6914.
- Deveraux, Q. et al. (1997) Nature 388:300.
- Deveraux, Q. and J. Reed (1999) Genes & Develop. 13:239.
- Herrera, B. et al. (2002) FEBS Letters 520:93.
Citations for Recombinant Human cIAP-1 (HIAP-2) 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.
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Citations: Showing 1 - 6
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DNA damage and S phase-dependent E2F1 stabilization requires the cIAP1 E3-ubiquitin ligase and is associated with K63-poly-ubiquitination on lysine 161/164 residues
Authors: V Glorian, J Allègre, J Berthelet, B Dumetier, PM Boutanquoi, N Droin, C Kayaci, J Cartier, S Gemble, G Marcion, D Gonzalez, R Boidot, C Garrido, O Michaud, E Solary, L Dubrez
Cell Death Dis, 2017-05-25;8(5):e2816.
Species: Bacteria - E. Coli, Human
Sample Types: Protein, Recombinant Protein
Applications: Bioassay -
USP11-dependent selective cIAP2 deubiquitylation and stabilization determine sensitivity to Smac mimetics.
Authors: Lee E, Seong D, Seo J, Jeong M, Lee H, Song J
Cell Death Differ, 2015-01-23;22(9):1463-76.
Species: Human
Sample Types: Recombinant Protein
Applications: Bioassay -
Cellular inhibitor of apoptosis (cIAP)-mediated ubiquitination of phosphofurin acidic cluster sorting protein 2 (PACS-2) negatively regulates tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) cytotoxicity.
Authors: Guicciardi, Maria Eu, Werneburg, Nathan W, Bronk, Steven F, Franke, Adrian, Yagita, Hideo, Thomas, Gary, Gores, Gregory
PLoS ONE, 2014-03-14;9(3):e92124.
Applications: Bioassay -
Role of melanoma inhibitor of apoptosis (ML-IAP) protein, a member of the baculoviral IAP repeat (BIR) domain family, in the regulation of C-RAF kinase and cell migration.
Authors: Oberoi-Khanuja, Tripat K, Karreman, Christia, Larisch, Sarit, Rapp, Ulf R, Rajalingam, Krishnar
J Biol Chem, 2012-06-18;287(34):28445-55.
Species: Human
Sample Types: Whole Cells
Applications: Bioassay -
Small molecules destabilize cIAP1 by activating auto-ubiquitylation.
Authors: Sekine K, Takubo K, Kikuchi R, Nishimoto M, Kitagawa M, Abe F, Nishikawa K, Tsuruo T, Naito M
J. Biol. Chem., 2008-01-29;283(14):8961-8.
Species: Human
Sample Types: Recombinant Protein
Applications: Surface Plasmon Resonance -
Enhanced ubiquitination of cytoskeletal proteins in pressure overloaded myocardium is accompanied by changes in specific E3 ligases.
Authors: Balasubramanian S, Mani S, Shiraishi H, Johnston RK, Yamane K, Willey CD, Cooper G, Tuxworth WJ, Kuppuswamy D
J. Mol. Cell. Cardiol., 2006-08-22;41(4):669-79.
Applications: Western Blot
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