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1. Product Manual OxiSelect Monoamine Oxidase Assay Kit Colorimetric Catalog Number XPX 5006 96 assays XPX 5006 5 5 x 96 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures CELL BIOLABS INC Creating Solutions for Life Science Research Introduction Monoamine Oxidases MAO are a collection of flavin adenine dinucleotide oxidoreductase enzymes found in the outer mitochondrial membrane MAOs catalyze the oxidative deamination of a variety of biogenic and xenobiotic amines MAOs include MAO A and MAO B which are two isoforms of the enzyme in mammals that have been distinguished based on localization inhibitor and substrate specificity Both MAO A and MAO B are omnipresent throughout brain liver and other tissues MAO A is predominantly found in the liver intestine brain and placenta whereas MAO B is found in the liver brain and platelets MAOs primary function is to regulate neurotransmitters such as dopamine noradrenaline or serotonin Dysfunction of MAO enzymes has been associated with many neurological disorders such as depression drug abuse migraines schizophrenia Attention Deficit Disorder ADD Parkinson s disease Alzheimer s disease as well as other disorders Cell Biolabs OxiSelect Monoamine Oxidase Assay Kit is a simple HTS compatible assay for measuring amine oxidase activity The assay can detect both MAO activity and semicarbizide sensitive amine oxidases SSAO In order to d2. A ms Inhibitor E BTyramine MAO B o Inhibitor D a m Benzylamine MAO A a Inhibitor O 0 2 E Benzylamine MAO B Inhibitor m Tyramine Only 0 1 0 Monoamine Oxidase A 50 pg mL Figure 2 Measurement of MAO A 50 ug mL of Monoamine Oxidase A was incubated with the MAO A Inhibitor Clorgyline or MAO B Inhibitor Pargyline according to the Assay Protocol These were subsequently incubated with the substrates Tyramine or Benzylamine within the Assay Working Solution for 45 minutes and read with a microplate reader at 540 nm Calculation of Results 1 Calculate the average absorbance values for every standard control and sample Subtract the average zero standard value from itself and all standard and sample values This is the corrected background absorbance If sample background control value is high subtract the sample background control value from the sample reading 2 Plot the corrected absorbance for the H2O standards against the final concentration of the hydrogen peroxide standards from Table 2 to determine the best slope uM See Figure 1 for an example standard curve 3 Use the standard curve to determine the hydrogen peroxide concentration generated by MAO 4 Determine the monoamine oxidase enzyme activity of the samples using the equation below Substitute the corrected fluorescence values for each sample Remember to account for dilution factors H202 generated MAO Activity Units L E x Sample dilution Reactio
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4. d that superoxide dismutase SOD be added to the reaction at a final concentration of 40 U mL Tatyana et al Ref 2 Avoid samples containing DTT or B mercaptoethanol since the probe is not stable in the presense of thiols above 10 uM Maintain pH between 7 and 8 for optimal working conditions as the probe is unstable at high pH gt 8 5 Preparation of Standard Curve To prepare the H20 standards first perform a 1 1000 dilution of the stock H20 in deionized water Prepare only enough for immediate use e g Add 5 uL of H20 to 4 995 mL deionized water This solution has a concentration of 8 8 mM Next further dilute the 8 8 mM in deionized water to prepare a 1 mM solution e g Add 114 uL of the 8 8 mM H20 solution to 886 uL deionized water Use this 1 mM H O solution to prepare standards in the concentration range of 0 uM 100 uM by further diluting in 1X Assay Buffer see Table 2 below H202 diluted solutions and standards should be prepared fresh each time the assay is tested Standard 1 mM H270 Standard 1X Assay Buffer Tubes uL uL H20 uM 1 100 900 100 2 500 of Tube 1 500 50 3 500 of Tube 2 500 25 4 500 of Tube 3 500 12 5 5 500 of Tube 4 500 6 25 6 500 of Tube 5 500 3 125 7 500 of Tube 6 500 1 56 9 0 500 0 Table 2 Preparation of H20 Standards Assay Protocol 1 Prepare and mix all reagents thoroughly before use Each sample including unknowns a
5. iscriminate between MAO A and MAO B the MAO A inhibitor Clorgyline and MAO B inhibitor Pargyline are included In addition two substrates Tyramine and Benzylamine Hydrochloride are included Tyramine will react with MAO A MAO B and SSAO while Benzylamine will react with MAO B and SSAO The two amine oxidase substrates are interchangeable in the assay and coupled with the provided MAO inhibitors can be used to determine MAO activity Applications for the kit include measuring amine oxidase in tissues blood samples and screening for amine oxidase inhibitors or substrates The kit has a detection sensitivity limit of 0 05 U L Each kit provides sufficient reagents to perform up to 96 assays including standard curve and unknown samples Assay Principle The OxiSelect Monoamine Oxidase Assay Kit is a simple and sensitive quantitative colorimetric assay for measuring amine oxidase activity in biological samples The assay can be utilized for both end point and kinetic measurements of Monoamine Oxidase MAO activity as well as semicarbazide sensitive amine oxidase SSAO Monoamine Oxidase reacts with its substrate and generates hydrogen peroxide H202 In the presence of HRP the Colorimetric Probe reacts with the H20 to produce a red pink colored product This product can be easily read by a microplate reader in the 540 570 nm range Absorbance values are proportional to the amine oxidase levels within the samples Unknown samples are dete
6. n Time minutes Note One unit of MAO catalyzes the formation of 1 umole of hydrogen peroxide per minute at 25 C 7 gt N CELL BIOLABS INC References 1 Hall D W R et al Biochem Pharmacol 1969 18 1447 1454 2 Tatyana V et al Neurochem 2001 79 266 3 Tipton K F et al Biochem J 1968 108 95 99 4 Youdim M B 1976 Preparation of Human Platelets In Monoamine Oxidase and It s Inhibition North Holland N Y 405 406 5 Youdim M B et al Methods Enzymol 1987 142 617 627 Warranty These products are warranted to perform as described in their labeling and in Cell Biolabs literature when used in accordance with their instructions THERE ARE NO WARRANTIES THAT EXTEND BEYOND THIS EXPRESSED WARRANTY AND CELL BIOLABS DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY OR WARRANTY OF FITNESS FOR PARTICULAR PURPOSE CELL BIOLABS sole obligation and purchaser s exclusive remedy for breach of this warranty shall be at the option of CELL BIOLABS to repair or replace the products In no event shall CELL BIOLABS be liable for any proximate incidental or consequential damages in connection with the products Contact Information Cell Biolabs Inc 7758 Arjons Drive San Diego CA 92126 Worldwide 1 858 271 6500 USA Toll Free 1 888 CBL 0505 E mail tech cellbiolabs com www cellbiolabs com 2015 Cell Biolabs Inc All rights reserved No part of these works may be reprodu
7. nd standards should be assayed in duplicate or triplicate 2 Add 50 uL of each sample H202 standard control or sample into an individual microtiter plate well 3 If assaying with MAO inhibitors add 5 uL of the 100 uM inhibitor to the appropriate MAO sample wells Add 5 uL Assay Buffer to the H202 standards and samples without inhibitor Mix the well contents thoroughly by pipetting or on a horizontal shaker and incubate 30 minutes at room temperature to allow the inhibitor to react with the enzyme Note The concentration of MAO A or MAO B inhibitors may be adjusted by the user 5 A o EAN CELL BIOLABS INC 4 Add 50 uL of Assay Working Solution to each well Mix the well contents thoroughly and incubate for 45 60 minutes at room temperature protected from light Note This assay is continuous not terminated and therefore may be measured at multiple time points to follow the kinetics of the reactions 5 Read the plate with a spectrophotometric microplate reader in the 540 570 nm range Example of Results The following figures demonstrate typical Monoamine Oxidase Colorimetric Assay results One should use the data below for reference only This data should not be used to interpret actual results 0 45 0 4 0 35 0 3 0 25 0 2 0 15 0 1 0 05 0 OD 540 nm Hydrogen Peroxide uM Figure 1 H O Standard Curve CELL BIOLABS INC gt o D 0 5 ETyramine MAO
8. olutions at 20 C MAO B Inhibitor Immediately before use prepare a 100 uM solution in 1X PBS e g Add 5 uL of the 20 mM inhibitor to 0 995 mL 1X PBS Vortex thoroughly Store solutions at 20 C Assay Working Solution Immediately before use prepare an Assay Working Solution using Table 1 below as a guide based on the number of assays needed Prepare by diluting the 200X Colorimetric Probe 1 100 100X Tyramine or 100X Benzylamine 1 50 and 100 U mL HRP to a final concentration of 0 2 U mL in 1X Assay Buffer The Assay Working Solution should be protected from light and used within 2 hours Prepare only enough for immediate use Note The Assay Working Solution will appear slightly pink in color This is normal background and should be subtracted from all absorbance values 1X Assay 100X Tyramine HRP 200X Total Volume Number of Assays Buffer mL or 100X uL Colorimetric of Assay in 96 well Plate Benzylamine Probe uL Working 50 pL well uL Solution mL 4 840 100 10 50 5 100 2 420 50 5 25 2 5 50 0 968 20 2 10 1 20 Table 1 Preparation of Assay Working Solution Preparation of Samples Note Samples should be assayed immediately or stored at 80 C prior to performing the assay Optimal experimental conditions for samples must be determined by the investigator The following recommendations are only guidelines and may be altered to optimize or complement the user s experimental design A se
9. r tube substrate for MAO B and SSAO MAO A Inhibitor Part No 50002C One 50 uL amber tube of 20 mM Clorgyline solution MAO B Inhibitor Part No 50003C One 50 uL amber tube of 20 mM Pargyline solution 10X Assay Buffer Part No 234403 One 25 mL bottle Materials Not Supplied ey Se eS E Distilled or deionized water 1X PBS for sample dilutions 10 uL to 1000 uL adjustable single channel micropipettes with disposable tips 50 uL to 300 uL adjustable multichannel micropipette with disposable tips Multichannel micropipette reservoir Spectrophotometric microplate reader capable of reading in the 540 570 nm absorbance range Reagents and equipment necessary for sample preparation Storage Upon receipt aliquot and store the HRP 100X Tyramine 100X Benzylamine MAO A Inhibitor and MAO B Inhibitor at 20 C The Colorimetric Probe is light sensitive and must be protected accordingly it may be stored at either 20 C or 80 C Avoid multiple freeze thaw cycles Store all remaining kit components at 4 C Preparation of Reagents Note All reagents must be brought to room temperature prior to use CELL BIOLABS INC yA o gt 1X Assay Buffer Dilute the stock 10X Assay Buffer 1 10 with deionized water for a 1X solution Stir or vortex to homogeneity MAO A Inhibitor Immediately before use prepare a 100 uM solution in 1X PBS e g Add 5 uL of the 20 mM inhibitor to 0 995 mL 1X PBS Vortex thoroughly Store s
10. rmined by comparison with a hydrogen peroxide standard curve Clorgyline a specific inhibitor of MAO A and Pargyline a specific inhibitor of MAO B are included in the kit to differentiate between the two enzymes The assay is simple sensitive and adaptable to high throughput testing Related Products 1 STA 320 OxiSelect Oxidative DNA Damage ELISA Kit 8 OHdG Quantitation 2 STA 341 OxiSelect Catalase Activity Assay Kit 3 STA 342 OxiSelect Intracellular ROS Assay Kit Green Fluorescence 4 STA 344 OxiSelect Hydrogen Peroxide Peroxidase Assay Kit Fluorometric 5 STA 347 OxiSelect In Vitro ROS RNS Assay Kit Green Fluorescence o gt CELL BIOLABS INC 6 STA 600 Phosphatidylcholine Assay Kit 7 STA 601 Sphingomyelin Assay Kit 8 9 STA 844 OxiSelect Hydrogen Peroxide Peroxidase Assay Kit Colorimetric STA 602 Acetylcholine Assay Kit Fluorometric 10 XPX 5000 OxiSelect Monoamine Oxidase Assay Kit Fluorometric Kit Components 1 wo gee e p O ye Se 96 well Protein Binding Plate Part No 231001 One 96 well strip plate 200X Colorimetric Probe Part No 239804 One 55 uL vial HRP Part No 234402 One 100 uL tube of 100 U mL solution in glycerol Hydrogen Peroxide Part No 234102 One 100 uL amber tube of an 8 82 M solution 100X Tyramine Part No 50001C One 100 uL amber tube substrate for MAO A MAO B and SSAO 100X Benzylamine Part No 50004C One 100 uL ambe
11. t of serial dilutions is recommended for samples to achieve optimal assay results and minimize possible interfering compounds Run proper controls as necessary Always run a standard curve with samples Platelets Prepare freshly drawn blood into polypropylene or polyethylene tubes with 1 10 volume of 1 5 EDTA 0 7 NaCl e g 20 mL blood 2 mL buffer The volume of blood depends on the experimental needs Centrifuge the tubes at 200 x g for 15 minutes at 4 C Carefully transfer the supernatant containing platelet rich plasma into clean tubes Avoid carry over of erythrocytes Centrifuge at 2000 x g for 10 minutes at 4 C Store the platelet pellet at 80 C until use Immediately before testing prepare cell lysate by sonication of the pellet in 1X PBS Youdim Ref 4 Cell or tissue mitochondrial fractions Isolate mitochondria using differential centrifugation for cell or tissue samples or by the method of choice Mitochondrial samples can be diluted in 1X PBS Notes CELL BIOLABS INC yA o gt All samples should be assayed immediately or stored at 80 C for up to 1 2 months Run proper controls as necessary Optimal experimental conditions for samples must be determined by the investigator Always run a standard curve with samples Samples with NADH concentrations above 10 uM and glutathione concentrations above 50 uM will oxidize the probe and could result in erroneous readings To minimize this interference it is recommende
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