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Caspase-8 (human), (recombinant)

Highly active caspase-8 for the study of enzyme regulation and kinetics
 
BML-SE172-5000 5000 U 404.00 USD
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Product Specification

Alternative Name:FLICE, Mch5, MACH
 
MW:18 + 11 kDa
 
Source:Produced in E. coli
 
UniProt ID:Q14790
 
Concentration:100 U/µl
 
Formulation:Liquid. In HEPES/NaOH, pH 7.4, 100mM sodium chloride, 0.5% CHAPS, 1mM EDTA, 10% glycerol and 10mM DTT.
 
Purity:≥90% (SDS-PAGE)
 
Specific Activity:One U=1 pmol/min using Ac-IETD-pNA (200mM; Prod. No. ALX-260-045) 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 the remaining, unused enzyme quickly by snap-freezing in a dry/ice ethanol bath or liquid nitrogen, if possible. Avoid repeated freeze/defrost cycles.
NOTE: When stored under the above conditions, this enzyme is stable at the concentration supplied, in its current storage buffer. Procedures such as dilution of the enzyme followed by refreezing, could lead to loss of activity.
 
Scientific Background:Caspase-8 is an initiator caspase activated in the death-inducing signaling complexes (DISCs) of the TNF receptor superfamily (e.g. TRAIL, FasL, TNFα receptors).
 
Protocol:Caspase-8 (FLICE/MACH/Mch5) Assay
Assay buffer
(50mM HEPES, pH 7.4, 100mM NaCl, 0.1% CHAPS, 1mM EDTA, 10% glycerol, 10mM DTT)
Caspase-8 (Prod. No. BML-SE172)
Dilute to 10 U/µl in assay buffer just before use.
Ac-IETD-pNA (Prod. No. ALX-260-045) colorimetric substrate
(400 µM stock solution in Assay buffer; Store at -20°C. Warm to assay temperature before use.)
To 5mg net peptide (MW=639) add 156µl DMSO, to prepare 50mM stock. Dilute 50mM stock to 400µM 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-8 (10U/µl) to each appropriate well. Include 2 blanks (5µl assay buffer rather than caspase-8).
3) To start reaction, add 50µl Ac-IETD-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 p-nitroaniline of 10,500 M-1 cm-1, and adjusting for pathlength of sample (~0.5cm for 100µl in well of a ½ volume, 96-well plate).
 
BML-SE172 SDS-PAGE
SDS PAGE Analysis: Lane 1: MW Marker, Lane 2: 1µg (Prod. No. BML-SE172).
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BML-SE172 SDS-PAGE

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For Research Use Only. Not for use in diagnostic procedures.
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