ab Cyclooxygenase 2 (COX2) Inhibitor Screening Kit (Fluorometric)

Transcription

1 Version 2 Last updated 2 March 2017 ab Cyclooxygenase 2 (COX2) Inhibitor Screening Kit (Fluorometric) For the rapid, sensitive and accurate screening of potential Cyclooxygenase 2 (COX2) inhibitors. This product is for research use only and is not intended for diagnostic use. Copyright 2017 Abcam. All rights reserved

2 Table of Contents 1. Overview 1 2. Protocol Summary 2 3. Precautions 3 4. Storage and Stability 3 5. Limitations 4 6. Materials Supplied 4 7. Materials Required, Not Supplied 5 8. Technical Hints 6 9. Reagent Preparation Sample Preparation Assay Procedure Calculations Typical Data Quick Assay Procedure Troubleshooting Notes 16 Copyright 2017 Abcam. All rights reserved

3 1. Overview Cyclooxygenase 2 (COX2) Inhibitor Screening Kit (Fluorometric) (ab211097) provides a rapid, simple, sensitive and reliable test suitable for high-throughput screening of COX2 inhibitors. The assay is based on the fluorometric detection of Prostaglandin G2 (PG2), the intermediate product generated by the COX enzyme. This simple and high-throughput adaptable assay kit can be used to screen, study or characterize potential inhibitors of COX2. COX + COX probe Arachidonic Acid PG2 Fluorescence (Ex/Em = 535/587 nm) COX + COX probe Arachidonic Acid PG2 Fluorescence + inhibitor (Ex/Em = 535/587 nm) Cyclooxygenase (COX, prostaglandin-endoperoxide synthase, PTGS, EC ) is an enzyme that is responsible for the formation of important biological mediators called prostanoids, including prostaglandins, prostacyclin and thromboxane. COX is the central enzyme in the biosynthetic pathway to prostanoids from arachidonic acid. There are two known isoenzymes: COX-1 and COX-2. COX-1 is constitutively expressed in many tissues and is the predominant form in gastric mucosa and in kidney. COX-2 is not expressed under normal conditions in most cells, but elevated levels are found during inflammation. Pharmacological inhibition of COX by nonsteroidal anti-inflammatory drugs (NSAID) can provide relief from the symptoms of inflammation and pain. ab COX2 Inhibitor Screening Kit 1

4 2. Protocol Summary Screening compound and controls preparation Reaction master mix preparation Add reaction master mix Measure fluorescence at Ex/Em (535/587 nm) in kinetic mode for 5 10 minutes at 25 C *For kinetic mode detection, incubation time given in this summary is for guidance only ab COX2 Inhibitor Screening Kit 2

5 3. Precautions Please read these instructions carefully prior to beginning the assay. All kit components have been formulated and quality control tested to function successfully as a kit. We understand that, occasionally, experimental protocols might need to be modified to meet unique experimental circumstances. However, we cannot guarantee the performance of the product outside the conditions detailed in this protocol booklet. Reagents should be treated as possible mutagens and should be handled with care and disposed of properly. Please review the Safety Datasheet (SDS) provided with the product for information on the specific components. Observe good laboratory practices. Gloves, lab coat, and protective eyewear should always be worn. Never pipette by mouth. Do not eat, drink or smoke in the laboratory areas. All biological materials should be treated as potentially hazardous and handled as such. They should be disposed of in accordance with established safety procedures. 4. Storage and Stability Store kit at -20 C in the dark immediately upon receipt. Kit has a storage time of 1 year from receipt, providing components have not been reconstituted. Refer to list of materials supplied for storage conditions of individual components. Observe the storage conditions for individual prepared components in the Materials Supplied section. Aliquot components in working volumes before storing at the recommended temperature. Note: Reconstituted components are stable for 2 months. ab COX2 Inhibitor Screening Kit 3

6 5. Limitations Assay kit intended for research use only. Not for use in diagnostic procedures. Do not mix or substitute reagents or materials from other kit lots or vendors. Kits are QC tested as a set of components and performance cannot be guaranteed if utilized separately or substituted. 6. Materials Supplied Item Quantity Storage temperatur e (before prep) Storage temperatur e (after prep) COX Assay Buffer 25 ml -20 C -20 C COX Probe (in DMSO) 200 µl -20 C -20 C COX Cofactor (in DMSO) 20 µl -20 C -20 C Arachidonic Acid (In EtOH) 50 µl -20 C -20 C NaOH 500 µl -20 C -20 C COX-2 Human Recombinant (100 U) Celecoxib, COX-2 inhibitor (in DMSO) 1 vial -20 C -20 C 100 µl -20 C -20 C ab COX2 Inhibitor Screening Kit 4

7 7. Materials Required, Not Supplied These materials are not included in the kit, but will be required to successfully perform this assay: Microplate reader capable of measuring fluorescence at Ex/Em = 535/587 nm Sterile MilliQ water or other type of double distilled water (ddh 2 O) Pipettes and pipette tips, including multi-channel pipette Assorted glassware for the preparation of reagents and buffer solutions Tubes for the preparation of reagents and buffer solutions 96 well plate with clear flat bottom, preferably white DMSO ab COX2 Inhibitor Screening Kit 5

8 8. Technical Hints This kit is sold based on number of tests. A test simply refers to a single assay well. The number of wells that contain sample, control or standard will vary by product. Review the protocol completely to confirm this kit meets your requirements. Please contact our Technical Support staff with any questions. Selected components in this kit are supplied in surplus amount to account for additional dilutions, evaporation, or instrumentation settings where higher volumes are required. They should be disposed of in accordance with established safety procedures. Avoid foaming or bubbles when mixing or reconstituting components. Avoid cross contamination of samples or reagents by changing tips between sample and reagent additions. Ensure plates are properly sealed or covered during incubation steps. Ensure all reagents and solutions are at the appropriate temperature before starting the assay. Make sure all necessary equipment is switched on and set at the appropriate temperature. ab COX2 Inhibitor Screening Kit 6

9 9. Reagent Preparation Briefly centrifuge small vials at low speed prior to opening. 9.1 COX Assay Buffer (25 ml): Ready to use as supplied. Equilibrate to room temperature before use. Store at -20 C. 9.2 COX Probe (in DMSO, 200 µl): Ready to use as supplied. Warm by placing in a 37ºC bath for 1 5 minutes to thaw DMSO solution before use. Note: DMSO tends to be solid when stored at -20ºC, even when left at room temperature, so it needs to melt for few minutes at 37ºC. Aliquot probe so that you have enough volume to perform the desired number of assays. Store at -20 C protected from light and moisture. Once the probe is thawed, use within two months. Use at room temperature. 9.3 COX Cofactor (in DMSO, 20 µl): Ready to use as supplied. Warm by placing in a 37ºC bath for 1 5 minutes to thaw DMSO solution before use. Note: DMSO tends to be solid when stored at -20ºC, even when left at room temperature, so it needs to melt for few minutes at 37ºC. Aliquot cofactor so that you have enough volume to perform the desired number of assays. Store at -20 C protected from light and moisture. Once the cofactor is thawed, use within two months. Use at room temperature. 9.4 Arachidonic Acid (In EtOH, 50 µl): Ready to use as supplied. Equilibrate to room temperature before use. Aliquot so that you have enough volume to perform the desired number of assays. Store at -20 C. 9.5 NaOH (500 µl): Ready to use as supplied. Equilibrate to room temperature before use. Aliquot so that you have enough volume to perform the desired number of assays. Store at -20 C. ab COX2 Inhibitor Screening Kit 7

10 9.6 COX-2, Human Recombinant (lyophilized, 100 U): Reconstitute in 110 µl of sterile ddh 2 O. Aliquot enzyme so that you have enough volume to perform the desired number of assays. Avoid repeated freeze/thaw. Store at -80 C. Use within two months. For short-term storage (~ 2 weeks), COX-2 can be stored at -20 C. Keep on ice while in use. 9.7 Celecoxib, COX-2 inhibitor (in DMSO, 100 µl): Ready to use as supplied. Warm by placing in a 37ºC bath for 1 5 minutes to thaw DMSO solution before use. Note: DMSO tends to be solid when stored at -20ºC, even when left at room temperature, so it needs to melt for few minutes at 37ºC. Aliquot inhibitor so that you have enough volume to perform the desired number of assays. Store at -20 C protected from light and moisture. Once the inhibitor is thawed, use within two months. Use at room temperature. ab COX2 Inhibitor Screening Kit 8

11 10. Sample Preparation Always prepare a fresh set of samples and controls for every use. Discard working standard dilutions after use as they do not store well Screening Compounds: Dissolve test compounds into proper solvent Dilute to 10X the desired test concentration with COX Assay Buffer before use. Note: We suggest using different concentrations of test compounds if effective concentration is unknown. ab COX2 Inhibitor Screening Kit 9

12 11. Assay Procedure Equilibrate all materials and prepared reagents to room temperature prior to use. We recommend that you assay all controls and samples in duplicate. Thaw COX-2 enzyme on ice immediately prior use. The is stable for at least ~30 minutes on ice, but it should not be left on ice for longer periods. Note: preferred final solvent concentration should not be more than 5% by volume. If solvent exceeds 5%, include solvent control to test the effect of the solvent on enzyme activity Set up Reaction wells: Sample compound wells (S) = 10 µl test compounds. Inhibitor Control wells (IC) = 2 µl Celecoxib + 8 µl COX Assay Buffer. Enzyme Control wells (EC) = 10 µl COX Assay Buffer. OPTIONAL: Solvent control (SC) = 10 µl solvent Prepare Reaction: Just before use, dilute COX Cofactor 1:200 by adding 2 µl COX Cofactor to 398 µl of COX Assay Buffer. Mix well by pipetting up and down. Note: diluted COX Cofactor is stable for 1 hour at RT. We do not recommend storing diluted COX Cofactor Just before use, prepare Arachidonic Acid solution: 5 µl of supplied Arachidonic Acid + 5 µl NaOH. Vortex briefly to mix Dilute Arachidonic Acid/NaOH solution 1:10 with 90 µl sterile ddh 2 O (or make as much as needed). Vortex briefly to mix. Make as much as needed. Note: diluted Arachidonic Acid/NaOH is stable for at least 1 hour on ice. We do not recommend storing diluted Arachidonic Acid/NaOH solution. ab COX2 Inhibitor Screening Kit 10

13 Prepare 80 µl of enzyme solution for each reaction. Mix enough reagents for the number of assays to be performed. Prepare a master mix to ensure consistency. Component COX Reaction Mix (µl) COX Assay Buffer 76 COX Probe 1 Diluted COX Cofactor (1:200 dilution) 2 COX-2 enzyme Add 80 µl of Reaction Mix into each well Add 10 µl of diluted Arachidonic Acid/NaOH solution into each well with a multi-channel pipette to initiate the reactions at the same time Measurement: Note: Preset the plate reader to avoid delay in measurement after addition of Arachidonic Acid/NaOH solution Measure immediately fluorescence at Ex/Em = 535/587 nm on a microplate reader in kinetic mode, for 5-10 minutes at 25 C protected from light. ab COX2 Inhibitor Screening Kit 11

14 12. Calculations Use only the linear rate for calculation Plot readings for each sample test compound (S), inhibitor control (IC) and enzyme control (EC) Draw the line of the best fit to construct the curve (most plate reader software or Excel can do this step). Calculate the trend line equation (use the equation that provides the most accurate fit) Choose two points (T1 and T2) in the linear range of the plot and obtain the corresponding values for the absorbance (RFU1 and RFU2) Calculate Slope for all samples (S), Enzyme Control (EC) and Inhibitor control (IC) as follows: ΔRFU/ΔT = (RFU2 RFU1)/ (T2 T1) 12.5 Calculate the % Relative Inhibition as follows: % Relative Inhibition = Slope of EC Slope of S Slope of EC x 100 Note: Irreversible inhibitors that inhibit HRV 3 C Protease activity completely at the tested concentration will have ΔA = 0 and thus % Relative Inhibition will be 100%. Note: If RFU of SC < RFU of EC = make a higher stock of test inhibitor, or dissolve the inhibitor in lower concentration of the solvent; or use a different solvent if possible. If RFU of S < RFU of EC = treat as 100% inhibition and further dilute the test inhibitor and repeat the assay. ab COX2 Inhibitor Screening Kit 12

15 13. Typical Data Data provided for demonstration purposes only. Figure 1. Typical inhibition curve of Cyclooxygenase 2 (COX-2) activity by COX-2 inhibitor celecoxib (IC 50 = 0.45 µm). Assay was performed following the kit protocol. ab COX2 Inhibitor Screening Kit 13

16 14. Quick Assay Procedure Note: this procedure is provided as a quick reference for experienced users. Follow the detailed procedure when performing the assay for the first time. Reconstitute enzyme (aliquot if necessary) and thaw components; get equipment ready. Prepare test compounds in suitable solvents; dilute if appropriate. Dilute COX Cofactor 1:200: 2 µl COX Cofactor µl of COX Assay Buffer. Just before use prepare AA/NaOH solution: 5 µl Arachidonic Acid + 5 µl NaOH Dilute AA/NaOH solution 1:10 times by adding 90 µl ddh 2 O. Prepare COX Reaction Mix for all wells to be set up (80 µl/well): Component COX Mix (µl) COX Assay Buffer 76 COX Probe 1 Diluted COX Cofactor (1:200) 2 COX-2 1 Set up plate as follows: Component Sample (S) (µl) Solvent control (SC) (µl) Enzyme Control (EC) (µl) Inhibitor Control (IC) (µl) Test Compound Solvent test compound Inhibitor Control Assay Buffer COX Reaction Mix Add 10 µl diluted Arachidonic Acid/NaOH solution Measure immediately at Ex/Em = 535/587 nm in kinetic mode, for 5-10 minutes at 25 C protected from light. ab COX2 Inhibitor Screening Kit 14

17 15. Troubleshooting Problem Reason Solution Assay not working Assay with erratic readings No fluorescence above background in inhibitor wells No inhibition seen in test compound wells Use of ice-cold buffer Plate read at incorrect wavelength Use of a different microplate Pipetting errors Air bubbles formed in well Inhibitor concentration is too high Inhibitor concentration is not high enough Compound is not an inhibitor Buffers must be at assay temperature Check the wavelength and filter settings of instrument Colorimetric: clear plates Fluorometric: black wells/clear bottom plates Luminometric: white wells/clear bottom plates Avoid pipetting small volumes (< 5 µl) and prepare a master mix whenever possible Pipette gently against the wall of the tubes Reduce concentration of inhibitor and re-do assay Increase concentration of inhibitor and re-do assay Use another compound for your test ab COX2 Inhibitor Screening Kit 15

18 16. Notes ab COX2 Inhibitor Screening Kit 16

19 ab COX2 Inhibitor Screening Kit 17

20 Technical Support Copyright 2017 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. All information / detail is correct at time of going to print. Austria wissenschaftlicherdienst@abcam.com France supportscientifique@abcam.com Germany wissenschaftlicherdienst@abcam.com Spain soportecientifico@abcam.com Switzerland technical@abcam.com Deutsch: Français: UK, EU and ROW technical@abcam.com +44(0) Canada ca.technical@abcam.com US and Latin America us.technical@abcam.com Asia Pacific hk.technical@abcam.com (852) China cn.technical@abcam.com Japan technical@abcam.co.jp +81-(0) Singapore sg.technical@abcam.com Australia au.technical@abcam.com +61-(0) New Zealand nz.technical@abcam.com +64-(0) Copyright 2017 Abcam. All rights reserved