ab83464 Catalase Assay Kit Instructions for Use For the rapid, sensitive and accurate measurement of Catalase activity in various samples. This product is for research use only and is not intended for diagnostic use.
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Table of Contents 1. Overview 3 2. Protocol Summary 3 3. Components and Storage 4 4. Assay Protocol 6 5. Data Analysis 9 6. Troubleshooting 11 2
1. Overview Catalase (EC 1.11.1.6) is a ubiquitous antioxidant enzyme that is present in nearly all living organisms. It functions to catalyze the decomposition of hydrogen peroxide (H 2 O 2 ) to water and oxygen. Abcam s Catalase Assay Kit provides a highly sensitive, simple, direct and HTS-ready assay for measuring Catalase activity in biological samples. In the assay, catalase first reacts with H 2 O 2 to produce water and oxygen, the unconverted H 2 O 2 reacts with OxiRed probe to produce a product, which can be measured at 570 nm (Colorimetric method) or at Ex/Em=535/587nm (fluorometric method). Catalase activity is reversely proportional to the signal. The kit detects high pico-unit of catalase in samples. 2. Protocol Summary Sample and Positive Control Preparation H 2 O 2 Standard Curve Preparation Add H 2 O 2 A Add Develop Mix Measure Optical Density 3
3. Components and Storage A. Kit Components Item Quantity Catalase Assay Buffer 25 ml OxiRed Probe (in DMSO) 200 µl HRP 1 vial H 2 O 2 (3%; 0.88M) 25 µl Stop Solution 1 ml Catalase Positive Control 1 vial * Store the kit at -20 C, protect from light. Warm the Assay Buffer to room temperature before use. Briefly centrifuge vials before opening. Keep samples, HRP and Catalase on ice during the assay. All these components are stable for 2 weeks at 4 C or for 1 month at -20 C after reconstitution. 4
Read the entire protocol before performing the assay. OxiRed PROBE: Briefly warm at 37 C for 1-2 min to completely melt the DMSO solution. Store at 20 C, protected from light. Use within two months. HRP SOLUTION: Dissolve with 220 μl Assay Buffer; it is sufficient for 100 assays. POSITIVE CONTROL SOLUTION: Dissolve positive control vial into 500 μl Assay Buffer. Aliquot 100 μl per vial, store at -20 C. B. Additional Materials Required Microcentrifuge Pipettes and pipette tips Fluorescent or colorimetric microplate reader 96 well plate Orbital shaker 5
4. Assay Protocol 1. Sample and Positive Control Preparations: a) Homogenize 0.1 gram tissues, or 10 6 Cells, or 0.2 ml Erythrocytes on ice in 0.2 ml cold Assay Buffer; Centrifuge at 10,000 x g for 15 min at 4 C; Collect the supernatant for assay, keep on ice. Liquid samples can be tested directly. Store samples at -80 C to assay later. b) Add 2-78 μl of samples or 1-10 μl Positive Control Solution into each well, and adjust volume to total 78 μl with Assay Buffer. Prepare sample High Control (HC) with the same amount of sample in separate wells then bring total volume to 78 μl with Assay Buffer. c) Add 10 μl of Stop Solution into the sample HC, mix and incubate at 25 C for 5 min to completely inhibit the catalase activity in samples as High Control. For unknown samples, we suggest testing several doses of your sample to ensure the readings are within the linear range. Note: Reducing agents in samples may interfere with the assay. Keep DTT or 2-mercaptoethanol below 5 μm. 6
2. H 2 O 2 Standard Curve Preparation: a) Dilute 5 μl of 0.88M H 2 O 2 into 215 μl dh 2 O to generate 20 mm H 2 O 2, then take 50 μl of the 20 mm H 2 O 2 and dilute into 0.95 ml dh 2 O to generate 1 mm H 2 O 2.* b) Add 0, 2, 4, 6, 8, 10 μl of 1 mm H 2 O 2 solution into 96-well plate to generate 0, 2, 4, 6, 8, 10 nmol/well H 2 O 2 standard. Bring the final volume to 90 μl with Assay Buffer. c) Add 10 μl Stop Solution into each well. For the fluorometric assay, dilute the standard H 2 O 2 10-fold for the standard curve (0-1 nmol range). * Note: Diluted H 2 O 2 is unstable, prepare fresh dilution each time. 3. Catalase Reaction: Add 12 μl fresh 1 mm H 2 O 2 into each well of both samples and sample HC to start the reaction, incubate at 25 C for 30 min, and then add 10 μl Stop Solution into each sample vial to stop the reaction. ** ** Note: High Control and Standard Curve wells already contain Stop Solution 7
4. Developer Mix: Mix enough reagents for the number of assays to be performed. For each well prepare a 50 μl Developer Mix containing: Assay Buffer 46 μl OxiRed Probe 2 μl HRP solution 2 μl Add 50 μl of the Developer Mix to each test samples, controls, and standards. Mix well and incubate at 25 C for 10 min. Add 10 μl Stop Solution and mix. Note: For low amounts of catalase, you can either increase the incubate time prior to adding the Stop Solution or use the fluorometric method. For the fluorometric method, decrease the 1 mm H 2 O 2 amount to1.5 μl and OxiRed Probe to 0.3 μl in the reaction; compensate the volume with Assay Buffer. 5. Measure OD 570nm in a plate reader. 8
5. Data Analysis Signal changes by catalase in sample is ΔA = AHC ASample. AHC is the reading of sample High Control, ASample is the reading of sample in 30 min. Plot the H 2 O 2 Standard Curve. Apply the ΔA to the H 2 O 2 standard curve to get B nmol of H 2 O 2 decomposed by catalase in 30 min reaction. Catalase activity can be calculated: Catalase B Activity = 30 x V x Sample Dilution Factor = nmol/min/ml = mu/ml Where: B is the decomposed H 2 O 2 amount (nmol) from H 2 O 2 Standard Curve. V is the pretreated sample volume (ml) added into the reaction well. 30 is the reaction time 30 min. Unit definition: One unit of catalase is the amount of catalase decomposes 1.0 μmol of H 2 O 2 per min at ph 4.5 at 25 C. 9
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6. Troubleshooting Problem Reason Solution Assay not working Unexpected results Assay buffer at wrong temperature Protocol step missed Plate read at incorrect wavelength Unsuitable microtiter plate for assay Measured at wrong wavelength Samples contain impeding substances Unsuitable sample type Sample readings are outside linear range Assay buffer must not be chilled - needs to be at RT Re-read and follow the protocol exactly Ensure you are using appropriate reader and filter settings (refer to datasheet) Fluorescence: Black plates (clear bottoms); Luminescence: White plates; Colorimetry: Clear plates. If critical, datasheet will indicate whether to use flat- or U-shaped wells Use appropriate reader and filter settings described in datasheet Troubleshoot and also consider deproteinizing samples Use recommended samples types as listed on the datasheet Concentrate/ dilute samples to be in linear range 11
Samples with inconsistent readings Lower/ Higher readings in samples and standards Unsuitable sample type Samples prepared in the wrong buffer Samples not deproteinized (if indicated on datasheet) Cell/ tissue samples not sufficiently homogenized Too many freezethaw cycles Samples contain impeding substances Samples are too old or incorrectly stored Not fully thawed kit components Out-of-date kit or incorrectly stored reagents Reagents sitting for extended periods on ice Incorrect incubation time/ temperature Incorrect amounts used Refer to datasheet for details about incompatible samples Use the assay buffer provided (or refer to datasheet for instructions) Use the 10kDa spin column (ab93349) Increase sonication time/ number of strokes with the Dounce homogenizer Aliquot samples to reduce the number of freeze-thaw cycles Troubleshoot and also consider deproteinizing samples Use freshly made samples and store at recommended temperature until use Wait for components to thaw completely and gently mix prior use Always check expiry date and store kit components as recommended on the datasheet Try to prepare a fresh reaction mix prior to each use Refer to datasheet for recommended incubation time and/ or temperature Check pipette is calibrated correctly (always use smallest volume pipette that can pipette entire volume) 12
Problem Reason Solution Standard curve is not linear Not fully thawed kit components Pipetting errors when setting up the standard curve Incorrect pipetting when preparing the reaction mix Air bubbles in wells Concentration of standard stock incorrect Errors in standard curve calculations Use of other reagents than those provided with the kit Wait for components to thaw completely and gently mix prior use Try not to pipette too small volumes Always prepare a master mix Air bubbles will interfere with readings; try to avoid producing air bubbles and always remove bubbles prior to reading plates Recheck datasheet for recommended concentrations of standard stocks Refer to datasheet and re-check the calculations Use fresh components from the same kit For further technical questions please do not hesitate to contact us by email (technical@abcam.com) or phone (select contact us on www.abcam.com for the phone number for your region). 13
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