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COX Activity
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Chemiluminescent Detection Kits |
| Format |
Catalog # |
Kit Insert |
MSDS |
Assay Layout |
| 96 Well Kit |
907-003 |
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| 100 Tube Kit |
907-011 |
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FOR RESEARCH PURPOSES ONLY, NOT FOR DIAGNOSTIC USE. |
 
Sensitivity: <0.0073 units/well or tube COX-1
Quantitate in 5-20 seconds
Direct Measurement of COX Enzyme Activity
Non-Radioactive
Simple and Rapid
Suitable for HTS Applications
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Cyclooxygenase (COX, also known as Prostaglandin G/H synthase) is a
membrane bound enzyme responsible for the oxidation of arachidonic acid
to Prostaglandin G2 (PGG2) and the subsequent
reduction of PGG2 to PGH2. The conversion is shown
below. These reactions are the first steps in the formation of a variety
of prostanoids. COX has been shown to be expressed in at least two
different isoforms: a constitutively expressed form, COX-I, and an
inducible form, COX-II. COX-I is thought to regulate a number of 'housekeeping'
functions, such as vascular hemostasis, renal blood flow, and
maintenance of glomerular function. Inflammation mediators such as
growth factors, cytokines and endotoxin induce COX-II expression in a
number of cellular systems. The effect of various nonsteroidal
anti-inflammatory drugs (NSAIDs) on the activity of COX-I and -II is an
area of considerable interest. Some methods to determine COX activity
involve procedures such as measuring uptake of oxygen using an oxygraph,
measuring the conversion of radioactive arachidonic acid, or measuring
the prostaglandins formed from PGH2 (such as determining PGE2
using immunoassays)+Q Most of these methods are complex, time consuming,
and are prone to interferences.
The Cyclooxygenase Reaction
Assay Designs' Cyclooxygenase Activity Kit uses a specific
chemiluminescent substrate to detect the peroxidative activity of COX
enzymes. After inhibition by NSAIDs, the direct residual activity of COX
is measured by addition of a proprietary luminescent substrate and
arachidonic acid. Light emission starts immediately and is directly
proportional to the COX activity in the sample. The chemiluminescent
signal is measured over 5 seconds. Note: In this insert, one Unit of
COX activity is defined as the amount of enzyme needed to consume 1
nmole of oxygen per minute at 37 °C.
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1. Prepare enzyme dilutions, buffers and Arachidonic Acid Stock
Solution.
2. Pipet buffer, hematin and COX-I or COX-II and inhibitor into
duplicate tubes or wells (except for the blanks and zero activity) and
incubate.
3. Simultaneously pipet or inject the Substrate and Acachidonic Acid
into the tubes or wells.
4. Immediately read in a suitable luminometer or chemiluminescent
detector for 5 seconds.
5. Calculate COX-I or COX-II concentrations from the Standard Curve.
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COX (907-003 &
907-011) |
| RANGE |
7.5-60 Units/mL
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| SAMPLE SIZE |
50 µL |
| SAMPLES PER 96
WELL KIT |
40 in duplicate
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| SENSITIVITY |
0.147 Units/mL COX-I
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| PRECISION |
3.1-7.0% RLU
Intra-assay (%CV) |
| SPECIES
SPECIFICITY |
non-specific
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The following commonly used substances were tested for
interference with the luminescent signal generated in the
Cyclooxygenase Activity Kit. At the concentrations listed, the
following changes in luminescent signal generation were
observed.
Substance |
Test Conc. |
Signal Change (%) |
| Ethanol |
5% |
6 |
| Methanol |
5% |
1 |
| Dimethyl sulfoxide (DMSO) |
10% |
1 |
| N, N-Dimethyl formamide (DMF) |
1% |
12.7 |
| Tween 20 |
0.1% |
5 |
| EDTA |
2.5 nM |
7.3 |
| Protein |
0.1% |
0 |
| Protease Inhibitors* |
0.1% |
3.3 |
| Tissue Culture Media |
100% |
14.8 |
| PBS |
100% |
15.9 |
| Sodium Azide |
0.1% |
5.5 |
| Gentamicin |
0.01% |
0.1 |
*Contains Peafabloc, pepstatin, leupeptin, E-64, bestatin, and
aprotinin |
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+32 (0) 16
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+32 (0) 16
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