Lipoxygenase Inhibitor Screening Assay Kit

Catalog No. 760700

3GENERAL INFORMATION

TABLE OF CONTENTS GENERAL INFORMATION 3 Materials Supplied

4 Precautions

4 If You Have Problems

4 Storage and Stability

4 Materials Needed but Not Supplied

INTRODUCTION 5 Background

5 About This Assay

PRE-ASSAY PREPARATION 6 Reagent Preparation

7 Sample Preparation

ASSAY PROTOCOL 8 Plate Set Up

9 Performing the Assay

ANALYSIS 10 Calculations

12 Performance Characteristics

12 Linearity of the Assay

RESOURCES 14 Interferences

16 Troubleshooting

16 References

17 Related Products

18 Warranty and Limitation of Remedy

19 Plate Template

20 Notes

GENERAL INFORMATION

Materials Supplied

Number Item Quantity

1 Assay Buffer (10X) 1 vial

2 Developing Reagent 1 1 vial

3 Developing Reagent 2 1 vial

4 15-Lipoxygenase (Standard) 1 vial

5 Arachidonic Acid (Substrate) 1 vial

6 Linoleic Acid (Substrate) 1 vial

7 KOH 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.

! WARNING: Not for human or animal disease diagnosis or therapeutic drug use.

4 GENERAL INFORMATION 5INTRODUCTION

PrecautionsPlease read these instructions carefully before beginning this assay. For research use only. Not for human or diagnostic use.

If You Have ProblemsTechnical Service Contact Information

Phone: 888-526-5351 (USA and Canada only) or 734-975-3888Fax: 734-971-3641E-Mail: techserv@caymanchem.comHours: M-F 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 StabilityStore the arachidonic and linoleic acid substrates (vials #5 and #6) at -20°C. The remaining components of the kit should be stored at 0-4°C and used before the expiration date indicated on the outside of the box.

Materials Needed But Not Supplied1. A plate reader capable of measuring absorbance between 490-500 nm.2. Adjustable pipettors and a repeat pipettor.3. A source of pure water. Glass distilled water or HPLC-grade water is acceptable.4. Hydrogen peroxide (420 µM).

INTRODUCTION

BackgroundLipoxygenases (LOs) are non-heme iron-containing dioxygenases that catalyze the addition of molecular oxygen to fatty acids containing a cis,cis-1,4-pentadiene system. The initial product of this reaction is a 4-hydroperoxy cis-trans-1,3-conjugated pentadienyl moiety within the unsaturated fatty acid.1,2 The three main LO enzymes are designated 5-, 12-, and 15-LO based on the position of the introduced hydroperoxide. Linoleate and arachidonate are the common substrates for LOs in plants and animals.

About This AssayCayman’s Lipoxygenase Inhibitor Screening Assay Kit detects and measures the hydroperoxides produced in the lipoxygenation reaction using a purified LO. The detection reaction is equally sensitive to hydroperoxides at various positions within the fatty acid, and will work with fatty acids of any carbon length. It is thus a general detection method for LO, and can be used to screen libraries of compounds for those which inhibit LO enzymes.

6 PRE-ASSAY PREPARATION 7PRE-ASSAY PREPARATION

PRE-ASSAY PREPARATION

Reagent Preparation1. Assay Buffer (10X) – (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 7.4) should be used for dilution of samples and the 15-LO standard prior to assaying. When stored at 4°C, this diluted Assay Buffer is stable for at least two months.

2. Developing Reagent 1 – (vial #2)The reagent is ready to use as supplied.

3. Developing Reagent 2 - (vial #3)The reagent is ready to use as supplied.

4. ChromogenPrepare the Chromogen prior to use by mixing equal volumes of Developing Reagent 1 (vial #2) and Developing Reagent 2 (vial #3) in a test tube and vortexing. The volume of Chromogen to be prepared is dependent on the number of wells assayed. Calculate 100 µl for each well. Use the Chromogen within one hour.

5. 15-Lipoxygenase (soybean) - (vial #4)A solution of 15-LO is supplied as a positive control. Transfer 10 µl of the supplied enzyme to another vial and dilute with 990 µl of assay buffer (dilute) prior to use, store on ice, and use within one hour. A 90 µl aliquot of the enzyme per well causes a final absorbance of approximately 0.19 under the standard assay conditions.

6. Arachidonic acid (substrate) - (vial #5)This vial contains a solution of arachidonic acid in ethanol and should be stored at -20°C when not being used. Transfer 25 µl of the supplied substrate to another vial, add 25 µl of KOH (vial #7), vortex, and dilute with 950 µl of HPLC-grade water to achieve a final concentration of 1 mM. Use the prepared arachidonic acid solution within 30 minutes. A 10 µl aliquot will yield a final concentration of 100 µM in the wells. NOTE: You can use either arachidonic or linoleic acid in the assay. You do not have to use both.

7. Linoleic acid (substrate) - (vial #6)This vial contains a solution of linoleic acid in ethanol and should be stored at -20°C when not being used. Transfer 25 µl of the supplied substrate to another vial, add 25 µl of KOH (vial #7), vortex, and dilute with 950 µl of HPLC-grade water to achieve a final concentration of 1 mM. Use the prepared linoleic acid solution within 30 minutes. A 10 µl aliquot will yield a final concentration of 100 µM in the wells. NOTE : You can use either arachidonic or linoleic acid in the assay. You do not have to use both.

8. KOH - (vial #7)This vial contains 0.1 M KOH. The reagent is ready to use as supplied.

Sample PreparationCell lysates and tissue homogenates contain peroxidases (e.g., glutathione peroxidase) that will reduce the lipid hydroperoxides generated in the assay, resulting in a very low signal. To achieve the most accurate results, we recommend screening purified LOs (5-, 12-, or 15-LO) with this assay. The sample must be free of particulates to avoid interferences in the absorbance measurement. Phosphates, EDTA, transition metal ions, thiols, and any endogenous LO inhibitors must be removed from the samples before performing the assay (extensive dialysis or concentrating and reconstituting in a Tris buffer several times will eliminate most of the interfering substances of small molecular size).If the samples are too dilute, they can be concentrated using a membrane filter with a molecular weight cut-off of 30,000 Da (such as an Amicon centrifuge concentrator).Cyclooxygenases will not be measured by this assay. If you are concerned that the activity seen in your sample is due to a cyclooxygenase (COX-1 or COX-2), then add a non-selective COX inhibitor (i.e., indomethacin, Cayman Chemical Catalog No. 70270) as a control. NOTE: Cayman’s 5-LO (human recombinant, Catalog No. 60402) will not work in the assay. Cayman’s 12-LO (porcine, Catalog No. 60300) will work in the assay if the supplied buffer is exchanged for a Tris Buffer. Cayman’s 15-LO (soybean P1, Catalog No. 60700) and 5-LO (potato, Catalog No. 60401) will work in the assay as supplied.

8 ASSAY PROTOCOL 9ASSAY PROTOCOL

General Information• Thefinalvolumeoftheassayis210μlinallthewells.• Itisnotnecessarytouseallthewellsontheplateatonetime.• Iftheappropriateinhibitordilutionisnotknown,itmaybenecessarytoassayatseveral

dilutions.• UsetheAssayBuffer(dilute)intheassay.• Itisrecommendedthatsamplesbeassayedatleastinduplicate(triplicatepreferred).• Thebackgroundabsorbance(absorbanceoftheblankwells)shouldbe<0.22.

Performing the Assay1. Blank Wells - add 100 µl of assay buffer to at least two wells.2. Positive Control Wells (15-LO standard) - add 90 µl 15-LO and 10 µl of assay buffer

to at least two wells.3. 100% Initial Activity Wells - add 90 µl of sample and 10 µl of solvent (the same

solvent used to dissolve the inhibitor) to two wells. The 100% initial activity wells should result in approximately 10 nmol/min/ml of activity.

4. Inhibitor Wells - add 90 µl of sample and 10 µl of inhibitor* to two wells.5. Initiate the reaction by adding 10 µl of substrate (either arachidonic or linoleic acid)

to all the wells. Place the 96-well plate on a shaker for at least five minutes.6. Add 100 µl of Chromogen to each well to stop enzyme catalysis and develop the

reaction. Cover with a plate cover and place the 96-well plate on a shaker for five minutes.

7. Remove the cover and read the absorbance at 490-500 nm using a plate reader.*Inhibitors can be dissolved in assay buffer (dilute), methanol, DMSO, or ethanol. The inhibitor should be added to the assay in a final volume of 10 µl before initiating with substrate. In the event that the appropriate concentration of inhibitor is completely unknown, we recommend that several dilutions of the inhibitor be made.

ASSAY PROTOCOL

Plate Set UpThere is no specific pattern for using the wells on the plate. However, it is necessary to have some wells (at least two) designated as non-enzymatic controls (blanks). The absorbance of these wells must be subtracted from the absorbance measured in the sample wells. We suggest that you have at least two wells designated as positive controls. A typical layout of samples to be measured in duplicate is shown in Figure 1. We also suggest you record the contents of each well on the template sheet provided (see page 19).

B - Blank+ - Positive Control* - 100% Initial Activity Samples1-45 - Inhibitor samples

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Figure 1. Sample plate format

Pipetting Hints

• It is recommended that a repeating pipettor be used to deliver substrate and Chromogen to the wells.

• Use different tips to pipette sample, substrate, and Chromogen. • Before pipetting each reagent, equilibrate the pipette tip in that reagent

(i.e., slowly fill the tip and gently expel the contents, repeat several times).• Donotexposethepipettetiptothereagent(s)alreadyinthewell.

10 ANALYSIS 11ANALYSIS

ANALYSIS

CalculationsDetermination of the Reaction Rate1. Subtract the absorbance of the Blanks from the absorbance of the samples and divide

by the length of incubation (five minutes).

A500 (sample) - A500 (blank)5 minutes

A500/min =

2. Use the following formula to calculate the LO activity. The reaction rate at 500 nm can be determined using the Chromogen extinction coefficient of 9.47 mM-1.** One unit of enzyme utilizes one µmol of arachidonic or linoleic acid per minute at 25°C.

Lipoxygenase activity = A500/min

9.47 mM-1x 0.21 ml

0.09 mlx sample dilution = µmol/min/ml

**The extinction coefficient has been adjusted for the path length of the solution in the well.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 5- and 15-LO inhibition by nordihydroguaiaretic acid (NDGA) are shown on page 11.

NDGA Concentration (µM)0 6 12 14 18

% In

itia

l 15-

LO A

ctiv

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Figure 2. Inhibition of soybean 15-lipoxygenase by NDGA (IC50 = 9 μM).

NDGA Concentration (�M)0 10 25 35

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O A

ctiv

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Figure 3. Inhibition of potato 5-lipoxygenase by NDGA (IC50 = 15 μM).

12 ANALYSIS 13ANALYSIS

Performance CharacteristicsSensitivity:Under the standardized conditions described in this booklet, samples containing LO activity between 1-10 nmol/min/ml can be assayed without further dilution or concentration. The assay will detect 0.5-5 nmol of lipid hydroperoxides.

Linearity of the AssayThe following graphs exhibit the linearity of the assay using soybean 15-LO.

15-Lipoxygenase Activity (nmol/min/ml)

0 2 4 6 8 10 12

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sorb

ance

(50

0 n

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Figure 4. Linearity of the assay using linoleic acid as the substrate.

15-Lipoxygenase Activity (nmol/min/ml)0 2 4 6 8 10 12

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sorb

ance

(50

0 n

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Figure 5. Linearity of the assay using arachidonic acid as the substrate.

NOTE: The same results are obtained with 5-LO (potato) and 12-LO (porcine leukocyte).

14 RESOURCES 15RESOURCES

RESOURCES

InterferencesCulture Media and BuffersAll buffers and media should be tested for high background absorbances before doing any experiments. If the initial background absorbances are higher than 0.22 absorbance units then the samples should be diluted in assay buffer (dilute) or HPLC-grade water before performing the assay. Phosphate, HEPES, and EDTA interfere with the Chromogen and will result in no enzyme activity. Tris, borate, and EGTA work fine in the assay. DMEM (Dulbecco’s Modified Eagles Medium) and MEM (Minimum Essential Medium Eagle) exhibit high background absorbances and should not be used in the assay. However, F-12 (Ham Nutrient Mixture) does not interfere with the assay.

Thiols and Transition Metal IonsSamples containing thiols (i.e., glutathione, cysteine, dithiothreitol, or 2-mercaptoethanol) and transition metal ions (i.e., Fe, Mn, or Cu) will exhibit high background absorbances and interfere with LO activity determination. Extensive dialysis will eliminate most of the interfering substances of small molecular size.

SolventsInhibitors can be dissolved in methanol, ethanol, or dimethyl sulfoxide. The inhibitor should be added to the assay in 10 µl.

InhibitorsLO inhibitors should be tested for assay interference by following the protocol outlined below:1. Blank Wells - add 100 µl of assay buffer (dilute) to at least two wells.2. Blank Wells plus Inhibitor Wells - add 90 µl of assay buffer (dilute) and 10 µl of

inhibitor to at least two wells.3. Hydrogen Peroxide Wells - add 90 µl of assay buffer (dilute) and 10 µl of 420 µM

hydrogen peroxide (not supplied in the kit) to at least two wells.4. Hydrogen Peroxide plus Inhibitor Wells - add 80 µl of assay buffer (dilute), 10 µl of

420 µM hydrogen peroxide, and 10 µl of inhibitor to at least two wells.5. Initiate the reaction by adding 10 µl of substrate (either arachidonic or linoleic acid)

to all the wells. Place the 96-well plate on a shaker for five minutes.6. Add 100 µl of Chromogen to each well and develop the reaction. Cover with a plate

cover and place the 96-well plate on a shaker for five minutes.7. Remove the cover and read the absorbance at 500 nm using a plate reader.

NOTE: The blank plus inhibitor wells should not exhibit an absorbance >0.22. If the absorbance is above 0.22, then try diluting with assay buffer (dilute) or solvent the inhibitor is dissolved in. The hydrogen peroxide wells and the hydrogen peroxide plus inhibitor wells should exhibit approximately the same absorbance. If the hydrogen peroxide plus inhibitor wells exhibit an absorbance higher or lower than the hydrogen peroxide wells, then the inhibitor is interfering with the assay. Try diluting the inhibitor with more assay buffer (dilute) or solvent.

16 RESOURCES 17RESOURCES

Troubleshooting

Problem Possible Causes Recommended Solutions

Eratic values; dispersion of duplicates/triplicates

A. Poor pipetting/techniqueB. Bubble in the well(s)

A. Carefully tap the side of the plate with your finger to remove bubbles

B. Be careful not to splash the contents of the wells

No color development A. Sample, substrate, or Chromogen was not added to the well(s); enzyme activity was too low

B. Something is interfering with the Chromogen.

Make sure to add all components to the wells and standardize the assay with the 15-LO standard; concentrate the sample so that the activity falls within the range of the assay; see the Interference section (on page 14) to confirm that the sample does not contain something that will effect the performance of the assay

High background abosorbance (>0.22)

There is something interfering with the assay

See the Interference section (on page 14)

References1. Gaffney, B.J. Lipoxygenases: Structural principles and spectroscopy. Annu. Rev.

Biophys. Biomol. Struct. 25, 431-459 (1996).2. Yamamoto, S. Mammalian lipoxygenases: Molecular structures and functions.

Biochim. Biophys. Acta 1128, 117-131 (1992).

Related ProductsArachidonic Acid - Cat. No. 90010 5,6-dehydro Arachidonic Acid - Cat. No. 90020 Caffeic Acid - Cat. No. 70602 Eicosatriynoic Acid - Cat. No. 90200 15(S)-HETE - Cat. No. 34720 Linoleic Acid - Cat. No. 90150 5-Lipoxygenase Polyclonal Antiserum - Cat. No. 160402 5-Lipoxygenase (potato) Screening Enzyme - Cat. No. 60401 12-Lipoxygenase (human) Polyclonal Antiserum - Cat. No. 160302 12-Lipoxygenase (murine leukocyte) Polyclonal Antiserum - Cat. No. 160304 12-Lipoxygenase (porcine leukocyte) - Cat. No. 60300 15-Lipoxygenase (rabbit reticulocyte) Polyclonal Antiserum - Cat. No. 160704 15-Lipoxygenase (soybean P1) - Cat. No. 60700 Nordihydroguaiaretic Acid - Cat. No. 70300 REV 5901 - Cat. No. 70600

18 RESOURCES 19RESOURCES

Warranty and Limitation of RemedyCayman 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.

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NOTES

This document is copyrighted. All rights are reserved. This document may not, in whole or part, be copied, photocopied, reproduced, translated, or reduced to any electronic medium or machine-readable form without prior consent, in writing, from Cayman Chemical Company.©01/22/2008, Cayman Chemical Company, Ann Arbor, MI, All rights reserved. Printed in U.S.A.

Lipoxygenase Inhibitor Screening Assay

Colorimetric assay for the screening of Lipoxygenase Inhibitors by measuring the Lipoxgenase activity in cell cultures

and tissue homogenates.

CM760700

96

For illustrative purposes only.

To perform the assay the instructions for use provided with the kit have to be used.

Distributed by:

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Instructions for Use

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Symbols Version 3.5 / 2008-10-01

REF Cat.-No.: / Kat.-Nr.: / No.- Cat.: / Cat.-No.: / N.º Cat.: / N.–Cat.: / Αριθµός-Κατ.:

LOT Lot-No.: / Chargen-Bez.: / No. Lot: / Lot-No.: / Lote N.º: / Lotto n.: / Αριθµός -Παραγωγή:

Use by: / Verwendbar bis: / Utiliser à: / Usado por: / Usar até: / Da utilizzare entro: / Χρησιµοποιείται από:

No. of Tests: / Kitgröße: / Nb. de Tests: / No. de Determ.: / N.º de Testes: / Quantità dei tests: / Αριθµός εξετάσεων:

CONC Concentrate / Konzentrat / Concentré / Concentrar / Concentrado / Concentrato / Συµπύκνωµα

LYO Lyophilized / Lyophilisat / Lyophilisé / Liofilizado / Liofilizado / Liofilizzato / Λυοφιλιασµένο

IVD In Vitro Diagnostic Medical Device. / In-vitro-Diagnostikum. / Appareil Médical pour Diagnostics In Vitro. / Dispositivo Médico para Diagnóstico In Vitro. / Equipamento Médico de Diagnóstico In Vitro. / Dispositivo Medico Diagnostico In vitro. / Ιατρική συσκευή για In-Vitro ∆ιάγνωση.

Evaluation kit. / Nur für Leistungsbewertungszwecke. / Kit pour évaluation. / Juego de Reactivos para Evaluació. / Kit de avaliação. / Kit di evaluazione. / Κιτ Αξιολόγησης.

Read instructions before use. / Arbeitsanleitung lesen. / Lire la fiche technique avant emploi. / Lea las instrucciones antes de usar. / Ler as instruções antes de usar. / Leggere le istruzioni prima dell’uso. / ∆ιαβάστε τις οδηγίες πριν την χρήση.

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