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Recombinant Human cIAP-2 (HIAP-1) Protein, CF

Catalog #: 817-P2
2 Citations Datasheet
Catalog # Availability Size / Price Qty
817-P2-050
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Recombinant Human cIAP-2 (HIAP-1) 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 25-1000 nM.
Optimal dilutions should be determined by each laboratory for each application.
Source
E. coli-derived human cIAP-2/HIAP-1 protein
ATVID 10-His tag SIEGRA Human cIAP-2
(Asn2-Ser604)
Accession # Q13489
N-terminus C-terminus
Accession #
Q13489
N-terminal Sequence
Analysis
Ala
Predicted Molecular Mass
71 kDa
SDS-PAGE
73 kDa, reducing conditions

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817-P2

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.

817-P2

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, -70 °C as supplied.
  • 3 months, -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
  • Recombinant Human cIAP‑2/HIAP‑1 (rhcIAP-2) (Catalog # 817-P2)
  • 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
  • Cell Extracts from Jurkat E6 wild type cells (see above protocol)
  • 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 rhcIAP-2 (MW: 71,000 Da) in Extraction Buffer. Make the following serial dilutions: 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 rhcIAP-2 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 30 °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 rhcIAP-2 by plotting normalized activity vs. reaction concentration of rhcIAP-2 (step 4) with Semi-Log Fitting [y = A + B * Log(x)].  Solve for x when y = 50.
  11. Normalized activity may be determined using the following equation:
    12.      % Normalized Activity = Sample (RFU/min) - Inactive Control** (RFU/min) x 100%
    Total Control (RFU/min)
Per Reaction:
  • rhcIAP-2 curve:  1000, 600, 200, 100, 20, 10, and 2 nM
  • Substrate: 50 μM

Background: cIAP-2/HIAP-1

cIAP-2 (also known as MIHC and HIAP-1) is a member of the inhibitor of apoptosis (IAP) family of proteins that inhibit the proteolytic activity of mature caspases. cIAP-2 has 3 BIR (baculovirus inhibitor of apoptosis) domains, a RING finger domain, and a caspase recruitment domain (CARD). cIAP-2 inhibits caspases through the direct interaction of its 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 HtrA2/Omi. cIAP-2 is reported to be cleaved by HtrA2/Omi.

References
  1. Roy, N. et al. (1997) EMBO J. 23:6914.
  2. Deveraux, Q. et al. (1997) Nature 388:300.
  3. Deveraux, Q. and J. Reed (1999) Genes & Develop. 13:239.
  4. Srinivasula, S.M. et al. (2003) J. Biol. Chem. 278:31469.
  5. Yang, Q-H. et al. (2003) Genes Dev. 17:1487.
Entrez Gene IDs
330 (Human)
Alternate Names
AIP1; API2; BIRC3 baculoviral IAP repeat containing 3; BIRC3; cIAP2; c-IAP2; cIAP-2; HAIP1; HIAP1; HIAP-1; MALT2; MIHC; RNF49

Citations for Recombinant Human cIAP-2 (HIAP-1) 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. 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
  2. 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

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