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Caspase-1 (human), (recombinant)

Highly active caspase essential for apoptosis.
 
BML-SE168-5000 5000 U 404.00 USD
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Product Specification

Alternative Name:Interleukin 1β converting enzyme, IL-1β converting enzyme, ICE
 
MW:~20 + 10kDa
 
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.
 
UniProt ID:P29466
 
Concentration:100 U/µl
 
Formulation:Liquid. In 50mM HEPES, pH 7.4, containing 100mM sodium chloride, 0.5% CHAPS, 1mM EDTA, 10% glycerol and 10mM DTT.
 
Purity:≥90% (SDS-PAGE)
 
Purity Detail:Purified by multi-step chromatography.
 
Specific Activity:One U=1 pmol/min, using Ac-YVAD-pNA (200µM; Prod. No. ALX-260-026) as substrate, at 30°C.
 
Application Notes:Useful tool to study enzyme regulation and kinetics, cleave target substrates, screen for inhibitors.
 
Shipping:Shipped on Dry Ice
 
Long Term Storage:-80°C
 
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.
 
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.
 
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). 
 
BML-SE168 SDS-PAGE
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BML-SE168 SDS-PAGE

Product Literature References

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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