Recombinant Human cIAP-2 (HIAP-1) Protein, CF Summary
Details of Functionality
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.
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
71 kDa. Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
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.
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-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.
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-2 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 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 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.
Normalized activity may be determined using the following equation:
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Human cIAP-2 (HIAP-1) Protein, CF
BIRC3 baculoviral IAP repeat containing 3
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.
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.
Srinivasula, S.M. et al. (2003) J. Biol. Chem. 278:31469.
The concentration calculator allows you to quickly calculate the volume, mass or concentration of your vial. Simply enter your mass, volume, or concentration values for your reagent and the calculator will determine the rest.
Review this Product
Be the first to review our Recombinant Human cIAP-2 (HIAP-1) Protein, CF and receive a gift card or discount.