OxiSelect™ Catalase Activity Assay Kit, Fluorometric, Trial Size
Contents
1. Product Manual OxiSelect Catalase Activity Assay Kit Fluorometric Trial Size Catalog Number STA 339 T 50 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures CELL BIOLABS INC Creating Solutions for Life Science Research Introduction Oxidative stress is a physiological condition where there is an imbalance between concentrations of reactive oxygen species ROS and antioxidants However excessive ROS accumulation will lead to cellular injury such as damage to DNA proteins and lipid membranes The cellular damage caused by ROS has been implicated in the development of many disease states such as cancer diabetes cardiovascular disease atherosclerosis and neurodegenerative diseases Under normal physiological conditions cellular ROS generation is counterbalanced by the action of cellular antioxidant enzymes and other redox molecules Because of their potential harmful effects excessive ROS must be promptly eliminated from the cells by this variety of antioxidant defense mechanisms Hydrogen peroxide is an ROS that is a toxic product of normal aerobic metabolism and pathogenic ROS production involving oxidase and superoxide dismutase reactions Hydrogen peroxide is poisonous to eukaryotic cells and in high doses can initiate oxidation of DNA lipids and proteins which can lead to mutagenesis and cell death The cellular damage caused by H202 has been implicated in the development of many pathological conditi2. OxiSelect HORAC Activity Assay NS re Sb Kit Components 1 ADHP Probe Part No 234401 T One 25 uL amber tube of a 10 mM solution in DMSO 2 HRP Part No 234402 T One 10 uL tube of a 100 U mL solution in glycerol 3 Hydrogen Peroxide Part No 234102 T One 20 uL amber tube of an 8 82 M solution 4 10X Assay Buffer Part No 234403 T Two 1 5 mL tubes 5 Catalase Standard Part No 234101 T One 10 uL amber tube of 600 000 Units mL Note One unit is defined as the amount of enzyme that will form 1 0 mg purpurogallin from pyrogallol in 20 seconds at pH 6 0 and 20 C Note One unit is defined as the amount of enzyme that will decompose 1 0 umole of hydrogen peroxide per minute at pH 7 0 and 25 C Materials Not Supplied Distilled or deionized water 1X PBS for sample dilutions 37 C incubator Multichannel micropipette reservoir 10 uL to 1000 uL adjustable single channel micropipettes with disposable tips 50 uL to 300 uL adjustable multichannel micropipette with disposable tips oe M Be WORN Standard 96 well fluorescence black microtiter plate and or black cell culture microplate A a CELL BIOLABS INC 8 Fluorescence microplate reader capable of reading excitation in the 530 570 nm range and emission in the 590 600 nm range Storage Upon receipt store the ADHP probe and HRP at 20 C ADHP is light sensitive and must be stored accordingly Avoid multiple freeze thaw cycles Store the remain
3. abs Inc All rights reserved No part of these works may be reproduced in any form without permissions in writing eo CELL BIOLABS INC J A
4. ce only This data should not be used to interpret actual results 1000 2000 3000 4000 5000 Catalase mU mL Figure 2 Catalase Activity Assay Standard Curve 6 CELL BIOLABS INC A e A References NOP YY SS Mohanty J G et al J Immunol Methods 1997 202 133 Mueller S et al Anal Biochem 1997 245 55 Tatyana V et al Neurochem 2001 79 266 Votyakova V Reynolds I Archives of Biochemistry and Biophysics 2004 431 138 144 Zhang J et al Antioxid Redox Signal 2001 3 493 504 Zhou M et al Anal Biochem 1997 253 162 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 2013 2014 Cell Biol
5. ing kit components at 4 C Preparation of Reagents Note All reagents must be brought to room temperature prior to use e 1X Assay Buffer Dilute the stock 10X Assay Buffer 1 10 with deionized water for a 1X solution Stir or vortex to homogeneity e ADHP HRP Working Solution Prepare an ADHP HRP Working Solution by adding ADHP to a final concentration of 100 uM and HRP to a final concentration of 0 4 U mL in 1X Assay Buffer e g Add 10 uL ADHP stock solution and 4 uL HRP stock solution to 986 uL 1X Assay Buffer This volume is enough for 20 assays The ADHP HRP Working Solution is stable for 1 day Prepare only enough for immediate applications Protect the Working Solution from light e Hydrogen Peroxide Working Solution Prepare a Hydrogen Peroxide Working Solution by diluting the stock concentration to 40 uM in 1X Assay Diluent To prepare the H20 first perform a 1 1000 dilution of the stock H202 in deionized water Use only enough for immediate applications e g Add 5 uL of H20 to 4 995 mL deionized water This solution has a concentration of 8 82 mM 8820 uM Use this 8 82 mM H2O gt solution to prepare a 40 uM H2O gt solution in 1X Assay Buffer e g Add 2 5 uL of the prepared 8 82 mM H20 solution to 0 55 mL 1X Assay Buffer Mix thoroughly This volume is enough for 20 assays The Hydrogen Peroxide Working Solution is stable for 1 day Prepare only enough for immediate use Preparation of Samples Notes e All sample
6. mL or tissues at 50 mg mL in 1X Assay Buffer or PBS Homogenize or sonicate the cells on ice Centrifuge to remove debris The lysate can be assayed undiluted or titrated in 1X Assay Buffer or PBS e Plasma or Urine To remove insoluble particles centrifuge at 10 000 rpm for 5 min The supernatant can be assayed directly or diluted in 1X Assay Buffer or PBS Preparation of Standard Curve Use only enough Catalase Standard as necessary for immediate applications Immediately prior to use vigorously vortex the Catalase Standard stock vial to obtain a homogenous suspension and immediately remove the desired amount It is recommended to pipette up and down several times prior to removing the suspension Prepare fresh standards by diluting the 600 000 Units mL Catalase Standard stock 1 150 in 1X Assay Buffer for a 4 000 Units mL solution eg Add 5 uL of Catalase Standard stock tube to 745 uL of 1X Assay Buffer Vortex thoroughly Further dilute this 4 000 Units mL solution 1 1000 in 1X Assay Buffer to prepare a 4000 mUnits mL solution eg Add 2 uL of Catalase Standard stock tube to 2 mL of 1X Assay Buffer Vortex thoroughly Use the 4000 mUnits mL solution to prepare a series of catalase standards according to Table below Note If crystals are visible upon preparation gently warm the standards at 30 C with vortexing Tubes 4000 mUnits mL 1X Assay Buffer Catalase Catalase Standard uL Concentration Dilution
7. ons such as aging asthma arthritis diabetes cardiovascular disease atherosclerosis Down s Syndrome and neurodegenerative diseases Catalase is an antioxidant enzyme omnipresent in mammalian and non mammaliian cells that destroys hydrogen peroxide by dismutation Eukaryotic catalases are heme enzymes found in the liver kidney and erythrocytes in high concentrations while the lowest concentrations are in the connective tissues The enzyme is concentrated in the peroxisome subcellular organelles Cell Biolabs OxiSelect Catalase Activity Assay Kit is a quantitative fluorometric assay for measuring catalase activity from biological samples The kit employs a simple sensitive homogeneous no wash and HTS compatible assay for measuring catalase activity in samples such as cell lysates plasma and tissue homogenates Catalase containing samples can be incubated in a known amount of hydrogen peroxide In the presence of H20 and horseradish peroxidase HRP non fluorescent ADHP 10 Acetyl 3 7 dihydroxyphenoxazine is oxidized to the highly fluorescent Resorufin ADHP is known as one of the most sensitive and stable fluorogenic probes for detecting H202 The probe can be utilized to monitor H20 release from cells or enzyme coupled reactions The kit has a detection sensitivity limit of at least 30 mU mL catalase This Trial Size kit provides sufficient reagents to perform up to 50 assays including standard curve and unknown samples Assay P
8. rinciple The OxiSelect Catalase Activity Assay Kit is a sensitive quantitative fluorometric assay for detecting catalase activity First catalase induces decomposition of H2O2 to produce water and oxygen O2 The rate of disintegration of hydrogen peroxide into water and oxygen is proportional to the concentration of catalase See Reaction 1 in Figure 1 Next in the presence of HRP ADHP reacts with the remaining H O gt in a 1 1 stoichiometry to produce highly fluorescent Resorufin As the catalase activity increases the Resorufin signal decreases The Resorufin product can be easily read by a fluorescence microplate reader with an excitation filter of 530 560 nm and an emission filter of 590 nm See Reaction 2 in Figure 1 Fluorescence values are proportional to the catalase levels within the samples The catalase content in unknown samples is determined by comparison with the predetermined catalase standard curve j CELL BIOLABS INC Catalase Reaction 1 2 HO _ gt 2H 0 O ka HRP Reaction 2 H20 Remaining ADHP Resorufin Fluorescence Figure 1 Catalase Reactions Related Products STA 341 OxiSelect Catalase Activity Assay Kit Colorimetric STA 342 OxiSelect ROS Activity Assay STA 343 OxiSelect Peroxide Detection Assay Colorimetric STA 344 OxiSelect Hydrogen Peroxide Peroxidase Detection Assay Fluorometric STA 345 OxiSelect ORAC Activity Assay STA 346
9. s 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 e A serial dilution will be necessary depending on the total H2O2 present Extremely high levels of H202 500 uM final concentration can lower the fluorescence because excess H202 can further oxidize the reaction product Resorufin to nonfluorescent product Resazurin e Samples with NADH concentrations above 10 uM and glutathione concentrations above 50 LM will oxidize the ADHP probe and could result in erroneous readings To minimize this interference it is recommended that superoxide dismutase SOD be added to the reaction at a final concentration of 40 U mL Tayana et al Ref 2 e Avoid samples containing DTT or B mercaptoethanol since Resorufin is not stable in the presense of thiols above 10 uM e Cell Culture Supernatant To remove insoluble particles centrifuge at 10 000 rpm for 5 min The supernatant can be assayed directly or diluted as necessary Prepare the catalase standard curve in the same non conditioned media Serum samples are not recommended since serum could possibly interfere with the assay 4 h ZN CELL BIOLABS INC ae Note Maintain pH between 7 and 8 for optimal working conditions as the ADHP is unstable at high pH gt 8 5 e Cell Lysates Resuspend cells at 1 2 x 10 cells
10. uL mUnits mL 1 400 0 4000 2 300 100 3000 3 200 200 2000 4 100 300 1000 5 50 350 500 6 25 375 250 7 12 5 387 5 125 8 6 3 393 7 62 5 9 3 1 396 9 31 3 10 0 400 0 Table 1 Preparation of Catalase Standards CELL BIOLABS INC aS Assay Protocol 1 Prepare and mix all reagents thoroughly before use Each cell sample including unknown and standard should be assayed in duplicate or triplicate 2 Add 25 uL of catalase standards controls and samples into individual microtiter plate wells Add 25 uL of the 40 uM Hydrogen Working Solution into each microplate well containing standards samples and controls Mix thoroughly Incubate the reaction mixture for 30 minutes at room temperature Add 50 uL of the ADHP HRP Working Solution to each well with standards controls and samples Mix the well contents thoroughly and incubate on a shaker for 30 minutes at 37 C and 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 Read the plate with a fluorescence microplate reader equipped for excitation in the 530 570 nm range and for emission in the 590 600 nm range Calculate the activity of catalase within samples by comparing the sample absorbance to the standard curve Example of Results The following figures demonstrate typical Catalase Activity Assay results One should use the data below for referen
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