Product Details
Alternative Name: | Interleukin 1β converting enzyme, IL-1β converting enzyme, ICE |
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MW: | ~20 + 10kDa |
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Source: | Produced in E. coli. cDNA encodes residues identical to Asn120-His404 (C-terminus) at Genbank Accession No. M87507, except for an Asp381 to Glu change, introduced to stabilize the enzyme against autoproteolysis. |
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UniProt ID: | P29466 |
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Formulation: | Liquid. In 50mM HEPES, pH 7.4, containing 100mM sodium chloride, 0.5% CHAPS, 1mM EDTA, 10% glycerol and 10mM DTT. |
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Purity: | ≥90% (SDS-PAGE) |
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Purity Detail: | Purified by multi-step chromatography. |
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Activity: | 100 U/µl |
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Specific Activity: | One U=1 pmol/min, using Ac-YVAD-pNA (200µM; Prod. No. ALX-260-026) as substrate, at 30°C. |
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Application Notes: | Useful tool to study enzyme regulation and kinetics, cleave target substrates, screen for inhibitors. |
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Shipping: | Shipped on Dry Ice |
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Long Term Storage: | -80°C |
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Use/Stability: | After initial defrost, aliquot product into individual tubes and refreeze at -80°C. Avoid repeated freeze/defrost cycles.The enzyme is stable on ice for the time typically required to set up an experiment (30-60 min.), but may lose activity with prolonged storage on ice. It is recommended that thawing and dilution of the enzyme be done within as short a time as possible before start of the assay. The remaining, undiluted and unused enzyme should be refrozen quickly by, for example, snap-freezing in a dry ice ethanol bath or liquid nitrogen. The enzyme is stable to at least 4 freeze/thaw cycles. |
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Scientific Background: | First of the caspases described, caspase-1 was subsequently found to be homologous to Ced-3, the C. elegans caspase essential for apoptosis. |
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Protocol: | Caspase-1 (ICE) Assay (Catalog #BML-SE168):
Assay buffer: (50mM HEPES, pH 7.4, 100mM NaCl, 0.1% CHAPS, 1mM EDTA, 10% glycerol, 10mM DTT)
Caspase-1 (BML-SE168): Dilute to 10 U/μl in assay buffer, just before use.
Ac-YVAD-pNA (ALX-260-026) colorimetric substrate: (400 μM stock solution in Assay buffer; Store at -20°C. Warm to assay temperature before use.) To 5 mg net peptide (MW=629) add 159 μl DMSO, to prepare 50 mM stock. Dilute 50mM stock to 400 μM with Assay Buffer.
Reaction Conditions:
1) Add 45μl Assay buffer into 1/2 volume microtiter plate. Allow to equilibrate to assay temperature.
2) Add 5μl of caspase-1 (10U/μl) to each appropriate well. Include 2 blanks (5μl assay buffer rather than caspase-1).
3) To start reaction, add 50μl Ac-YVAD-pNA substrate (400μM in assay buffer). Final substrate concentration=200μM.
4) Continuously monitor A405nm.
5) Data analysis: Graph OD vs time and determine slope over the linear portion of the curve. Convert rates in OD/min to substrate/min using an extinction coefficient for pnitroaniline of 10,500M-1 cm-1, and adjusting for pathlength of sample (~0.5cm for 100μl in well of a ½ volume, 96-well plate). |
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Regulatory Status: | RUO - Research Use Only |
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Product Literature References
A small molecule inhibitor of caspase-1 inhibits NLRP3 inflammasome activation and pyroptosis to alleviate gouty inflammation: D.Y. Cao, et al.; Immunol. Lett.
244, 28 (2022),
Abstract;
The colonic pathogen Entamoeba histolytica activates caspase-4/1 that cleaves the pore-forming protein gasdermin D to regulate IL-1β secretion: S. Wang, et al.; PLoS Pathog.
18, e1010415 (2022),
Abstract;
An Essential Role for SHARPIN in the Regulation of Caspase 1 Activity in Sepsis: M.V. Nastase, et al.; Am. J. Pathol.
186, 1206 (2016),
Abstract;
iGLuc: a luciferase-based inflammasome and protease activity reporter: E. Bartok, et al.; Nat. Methods
10, 147 (2013),
Application(s): Recombinant Proteolysis,
Abstract;
Full Text
The Yersinia virulence effector YopM binds caspase-1 to arrest inflammasome assembly and processing: C.N. LaRock, et al.; Cell Host Microbe
12, 799 (2012),
Application(s): Incubation,
Abstract;
Full Text
Antiapoptotic mechanism of HIV protease inhibitors: preventing mitochondrial transmembrane potential loss: N. Barbara, et al.; Blood
98, 1078 (2001),
Abstract;
Full Text
Preparation of an autolysis-resistant interleukin-1 beta converting enzyme mutant: L.C. Dang, et al.; Biochemistry
35, 14910 (1996),
Abstract;
Interleukin-1 beta converting enzyme: N.A. Thornberry; Methods Enzymol.
244, 615 (1994),
Abstract;
The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1β-converting enzyme: J. Yuan, et al.; Cell
75, 641 (1993),
Abstract;
A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes: N.A. Thornberry, et al.; Nature
356, 768 (1992),
Abstract;
Molecular cloning of the interleukin-1 converting enzyme: D.P. Cerretti, et al.; Science
256, 97 (1992),
Abstract;
General Literature References
Preparation of an autolysis-resistant interleukin-1 beta converting enzyme mutant.: L.C. Dang et al.; Biochemistry 35, 14910 (1996),
Interleukin-1 beta converting enzyme.: N.A. Thornberry; Methods Enzymol. 244, 615 (1994),
The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1 beta-converting enzyme.: J. Yuan et al.; Cell 75, 641 (1993),
A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes.: N.A. Thornberry et al.; Nature 356, 768 (1992),
Molecular cloning of the interleukin-1 beta converting enzyme.: D.P. Cerretti et al.; Science 256, 97 (1992),
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