Chemiluminescent COX (ovine) Inhibitor Screening Assay Kit Catalog No. 760101
TABLE OF CONTENTS Contents of the Kit ...................................................................................................................................................................................2 Precautions................................................................................................................................................................................................2 Warranty and Limitation of Remedy.........................................................................................................................................................2 If You Have Problems ...............................................................................................................................................................................2 Storage and Stability .................................................................................................................................................................................2 Additional Items Required ........................................................................................................................................................................2 About this Assay........................................................................................................................................................................................3 Pre-Assay Preparation................................................................................................................................................................................4 Performing the Assay.................................................................................................................................................................................5 Calculations ..............................................................................................................................................................................................6 Interferences..............................................................................................................................................................................................7 Troubleshooting ........................................................................................................................................................................................7 References .................................................................................................................................................................................................7 Related Products .......................................................................................................................................................................................7 Plate Template...........................................................................................................................................................................................8 Notes ........................................................................................................................................................................................................8
1
CONTENTS OF THE KIT Number
Item
Quantity
1
Assay Buffer (10X)
1 vial
2
Heme
1 vial
3
COX-1 (ovine)
1 vial
4
COX-2 (ovine)
1 vial
5
Arachidonic Acid
1 vial
6
Potassium Hydroxide
1 vial
7
Chemiluminescent Substrate
1 vial
8
96 Well Plate
1 plate
9
Plate Cover
1 cover
If any of the items listed above are damaged or missing, please contact our Customer Service department at (800) 364-9897 or (734) 975-3999. We cannot accept any returns without prior authorization.
PRECAUTIONS • •
Please read these instructions carefully before beginning this assay. For research use only. Not for human or diagnostic use.
WARRANTY AND LIMITATION OF REMEDY Cayman Chemical Company makes no warranty or guarantee of any kind, whether written or oral, expressed or implied, including without limitation, any warranty of fitness for a particular purpose, suitability and merchantability, which extends beyond the description of the chemicals hereof. Cayman warrants only to the original customer that the material will meet our specifications at the time of delivery. Cayman will carry out its delivery obligations with due care and skill. Thus, in no event will Cayman have any obligation or liability, whether in tort (including negligence) or in contract, for any direct, indirect, incidental or consequential damages, even if Cayman is informed about their possible existence. This limitation of liability does not apply in the case of intentional acts or negligence of Cayman, its directors or its employees. Buyer’s exclusive remedy and Cayman’s sole liability hereunder shall be limited to a refund of the purchase price, or at Cayman’s option, the replacement, at no cost to Buyer, of all material that does not meet our specifications. Said refund or replacement is conditioned on Buyer giving written notice to Cayman within thirty (30) days after arrival of the material at its destination. Failure of Buyer to give said notice within thirty (30) days shall constitute a waiver by Buyer of all claims hereunder with respect to said material.
For further details, please refer to our Warranty and Limitation of Remedy located on our website and in our catalog.
IF YOU HAVE PROBLEMS Our technical support staff may be reached by phone (800-364-9897, 734-975-3888), fax (734-971-3641), or E-Mail (
[email protected]) Monday through Friday 8:00 AM to 5:30 PM EST. In order for our staff to assist you quickly and efficiently, please be ready to supply the lot number of the kit (found on the outside of the box).
STORAGE AND STABILITY This kit will perform as specified if stored at -80°C and used before the expiration date indicated on the outside of the box.
ADDITIONAL ITEMS REQUIRED 1. 2. 3.
A microplate Luminometer with one or two syringe dispensers. Adjustable pipettors and a repeat pipettor. A source of pure water. Glass distilled water or HPLC-grade water is acceptable.
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ABOUT THIS ASSAY Cyclooxygenase (also called Prostaglandin H Synthase or PGHS) catalyzes the first step in the biosynthesis of prostaglandins, thromboxanes, and prostacyclins; the conversion of arachidonic acid to prostaglandin H2 as illustrated in Figure 1. Until recently, only one form of cyclooxygenase had been characterized, cyclooxygenase-1 (COX-1). COX-1 is constitutively expressed in variety of cell types and is involved in normal cellular homeostasis. The induction of cyclooxygenase expression by a variety of stimuli such as phorbol esters, lipopolysaccharides, and cytokines led to the hypothesis that the inducible form of cyclooxygenase, cyclooxygenase-2 (COX-2), is responsible for the biosynthesis of prostaglandins under acute inflammatory conditions.1 This inducible COX-2 is believed to be the target enzyme for the anti-inflammatory activity of nonsteroidal anti-inflammatory drugs. Cyclooxygenase enzymes contain both cyclooxygenase and peroxidase activities. The Cayman Chemical Chemiluminescent COX (ovine) Inhibitor Screening Assay utilizes the heme-catalyzed hydroperoxidase activity of ovine cyclooxygenases to generate luminescence in the presence of a luminol-type cyclic naphthalene hydrazide and the substrate arachidonic acid.2 Arachidonate-induced luminescence was shown to be an index of real-time catalytic activity and demonstrated the turnover inactivation of the enzyme.2 Inhibition of cyclooxygenase activity, measured by luminescence, by a variety of selective and nonselective inhibitors showed potencies similar to those observed with other in vitro and whole cell methods.2 The Chemiluminescent COX assay includes both ovine COX-1 and COX-2 enzymes in order to screen isozyme-specific inhibitors. The Cayman Chemiluminescent COX assay is a time saving tool for screening vast numbers of inhibitors. COOH
Arachidonic Acid
Cyclooxygenase activity
COOH
O O
Prostaglandin G2
OOH
Peroxidase activity
COOH
O O
Prostaglandin H2
OH
Reduction or Non-specific decomposition
OH
O
HO
Prostaglandin E2
COOH
COOH HO
O OH
other products
OH
COOH
OH
OH
Prostaglandin D2
Prostaglandin F2α
Figure 1. Conversion of arachidonic acid.
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PRE-ASSAY PREPARATION Preparation of reagents 1.
Assay Buffer - (vial #1) Dilute 3 ml of Assay Buffer concentrate with 27 ml of HPLC-grade water. This final Assay Buffer (0.1 M Tris-HCl, pH 8) should be used for dilution of heme and arachidonic acid prior to assaying. When stored at 4°C, this diluted Assay Buffer is stable for at least two months.
2.
Heme - (vial #2) This vial contains a solution of heme in dimethyl sulfoxide. Dilute 58 µl of heme with 942 µl of Assay Buffer (dilute) prior to use. This diluted heme is stable for 12 hours at room temperature.
3.
COX-1 (ovine)- (vial #3) This vial contains a solution of ovine COX-1. To avoid repeated freezing and thawing, the COX-1 should be aliquoted into several small vials and stored at -80°C. Dilute 30 µl of enzyme with 570 µl of assay buffer (dilute) and store on ice. This is enough enzyme to do 60 wells. Scale the amount up if assaying more wells. The diluted enzyme is stable for one hour. There is enough COX-1 supplied to do 96 wells. NOTE: You will need 10 µl of enzyme per well.
4.
COX-2 (ovine) - (vial #4) This vial contains a solution of ovine COX-2. To avoid repeated freezing and thawing, the COX-2 should be aliquoted into several small vials and stored at -80°C. Dilute 30 µl of enzyme with 570 µl of assay buffer (dilute) and store on ice. This is enough enzyme to do 60 wells. Scale the amount up if assaying more wells. The diluted enzyme is stable for one hour. There is enough COX-2 supplied to do 96 wells. NOTE: You will need 10 µl of enzyme per well.
5.
Arachidonic acid (Substrate) - (vial #5) This vial contains a solution of arachidonic acid in ethanol and should be stored at -80°C when not being used. Transfer 100 µl of the supplied substrate to another vial, add 100 µl of KOH (vial #6), vortex, and dilute with 9.8 ml of Assay Buffer (dilute) to achieve a final concentration of 116 µM. Use the prepared arachidonic acid solution within 15 minutes. A 50 µl aliquot will yield a final concentration of 20 µM in the wells.
6.
KOH - (vial #6) This vial contains 0.1 M KOH. The reagent is ready to use as supplied.
7.
Chemiluminescent Substrate - (vial #7) This vial contains a naphthalene hydrazide solution. The reagent is ready to use as supplied.
Plate configuration There is no specific pattern for using the wells on the plate. However, it is necessary to have three wells designated as non-enzymatic or background wells. The relative luminescent units (RLU) of these wells must be subtracted from the RLUs measured in the COX wells. We suggest that each COX sample be assayed in triplicate. We suggest you record the contents of each well on the template sheet provided on page 8. PIPETTING HINTS •
It is recommended that a repeating pipettor be used to deliver assay buffer (dilute), enzyme, and heme to the wells.
•
Use different tips to pipet enzymes, heme, and assay buffer (dilute).
•
Before pipetting each reagent, equilibrate the pipet tip in that reagent (i.e., fill the tip and expel the contents; repeat several times).
•
Do not expose the pipet tip to the reagent(s) already in the well.
•
It is recommend to have the luminometer dispense both the chemiluminescent substrate and the arachidonic acid (a Luminometer with two syringes). If the Luminometer has only one dispenser, then pipet the chemiluminescent substrate into the wells, with a repeating pipettor, just before putting the plate into the Luminometer. 4
PERFORMING THE ASSAY •
The final volume of the assay is 290 µl in all the wells.
•
The assay is performed at 25°C.
•
You do not have to use both enzymes. You can use either COX-1 or COX-2 for the study.
•
If the appropriate inhibitor dilution is not known, it may be necessary to assay at several dilutions.
•
Use the Assay Buffer (dilute) in the assay.
•
We recommend assaying samples in triplicate.
•
It is recommended to have the Luminometer dispense both the chemiluminescent substrate and the arachidonic acid (a Luminometer with two syringes).
•
Prime the first Luminometer dispenser with the chemiluminescent substrate, and the second with the arachidonic acid solution.
•
It is recommended to do only two rows at a time so as to control the length of the incubation more accurately.
1.
Background Wells - add 10 µl of heme, 10 µl of Assay Buffer, and 10 µl of solvent (which ever you dissolved your inhibitor in) to three wells.
2.
100% Initial Activity Wells - add 10 µl of heme, 10 µl of Enzyme (either COX-1 or COX-2), and 10 µl of solvent (which ever you dissolved your inhibitor in) to three wells.
3.
Inhibitor Wells - add 10 µl of heme, 10 µl of Enzyme (either COX-1 or COX-2), and 10 µl of inhibitor* to three wells.
4.
Add 200 µl of Assay Buffer to all the wells you are using. NOTE: If your Luminometer has only one syringe dispenser then add 10 µl of chemiluminescent substrate using a repeat pipettor. DO NOT perform this step if your luminometer is dispensing the chemiluminescent substrate.
5.
Insert the plate into the Luminometer and have the Luminometer dispense 10 µl of the chemiluminescent substrate and then immediately dispense 50 µl of arachidonic acid to all the wells you are using.
6.
Immediately read for 10 seconds per well.
7.
Repeat steps 2-6 for another two rows.
*Inhibitors can be dissolved in methanol, dimethyl sulfoxide, or ethanol. In the event that the appropriate concentration of inhibitor is completely unknown, we recommend that several dilutions of the inhibitor be made.
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CALCULATIONS 1.
Determine the average Relative Luminescent Units (RLU) of all the samples.
2.
Subtract the RLU of the background wells from the RLUs of the 100% Initial Activity and the Inhibitor wells.
3.
Subtract each Inhibitor Sample from the 100% Initial Activity Sample, then divide by the 100% Initial Activity Sample, and multiply by 100 to give the percent inhibition.
4.
Either graph the Percent Inhibition or Percent Initial Activity by the Inhibitor Concentration to determine the IC50 value (concentration at which there was 50% inhibition). Examples of ovine COX-1 and COX-2 inhibition by indomethacin are shown below. ���
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Graph 1. Inhibition of ovine COX-1 by indomethacin (IC50 = 0.1 µM) 100
% Initial Activity
80
60
40
20
0 0
3
6
9
Indomethacin (�M)
12
15
Graph 2. Inhibition of ovine COX-2 by indomethacin (IC50 = 6 µM)
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INTERFERENCES Antioxidants Any antioxidant will interfere with the assay and will appear to be a cyclooxygenase inhibitor. Resveratrol is an antioxidant, as well as, a selective inhibitor of COX-1.3 Using this assay, Resveratrol will also appear to be a COX-2 inhibitor. If the inhibitor you are assaying is also an antioxidant, it is recommended that you use one of Cayman’s COX Inhibitor Screening Assays which utilizes an EIA detection (Catalog No. 560101 or 560121). Solvents Methanol, dimethyl sulfoxide, and ethanol have no effect on cyclooxygenase activity. Cyclooxygenase inhibitors can be dissolved in any of the above solvents and should be added to the assay in 10 µl.
TROUBLESHOOTING Problem: Erratic values; dispersion of triplicates. Causes: Poor pipetting/technique. Problem: No luminescence. Cause: Enzyme, arachidonic acid, or chemiluminescent substrate was not added to the well(s). -or- Enzyme activity was too low. -orSomething is interfering with the assay. Solution: Make sure to add all components to the wells and check to make sure the Luminometer is dispensing correctly. -or- Reduce the concentration of the inhibitor. -or- Make sure you are not adding an antioxidant to the assay. Problem: No inhibition seen with inhibitor. Cause: The inhibitor concentration is not high enough or the compound is not an inhibitor of that enzyme. Solution: Increase the inhibitor concentration and re-assay.
REFERENCES 1. 2. 3.
Xie, W., Chipman, J.G., Robertson, D.L., et al. Expression of a mitogen-responsive gene encoding prostaglandin synthase is regulated by mRNA splicing. Proc. Nat. Acad. Sci. USA 88, 2692-2696 (1991). Forghani, F., Ouellet, M., Keen, S., et al. Analysis of prostaglandin G/H synthase-2 inhibition using peroxidase-induced luminol luminescence. Anal. Biochem. 264, 216-221 (1998). Jang, M., Cai, L., Udeani, G.O., et al. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science 275, 218-220 (1997).
RELATED PRODUCTS COX-1 (ovine) - Cat. No. 60100 • COX-2 (ovine) - Cat. No. 60120 • (±)-Flurbiprofen - Cat. No. 70250 • Aspirin - Cat. No. 70260 • Indomethacin - Cat. No. 70270 • (±)-Ibuprofen - Cat. No. 70280 • APHS - Cat. No. 70330 • Ebselen - Cat. No. 70530 • NS-398 - Cat. No. 70590 • 6-methoxy Naphthalene Acetic Acid - Cat. No. 70620 • Nimesulide - Cat. No. 70640 • Niflumic Acid - Cat. No. 70650 • Valeryl Salicylate - Cat. No. 70670 • Resveratrol - Cat. No. 70675 • Diclofenac (sodium salt) - Cat. No. 70680 • Arachidonic Acid - Cat. No. 90010 • COX (ovine) Inhibitor Screening Assay - Cat. No. 560101 • Colorimetric COX (ovine) Inhibitor Screening Assay - Cat. No. 760111 • COX Activity Assay - Cat. No. 760151
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PLATE TEMPLATE
1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H NOTES
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