ab102500 Hydrogen Peroxide Assay Kit
Instructions for Use For the rapid, sensitive and accurate measurement of Hydrogen Peroxide 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
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2.
Protocol Summary
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3.
Components and Storage
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4.
Assay Protocol
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5.
Data Analysis
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6.
Troubleshooting
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1. Overview Hydrogen Peroxide (H2O2) is a reactive oxygen metabolic by-product that serves as a key regulator for a number of oxidative stressrelated states. Functioning through NF-κB and other factors, hydroperoxide-mediated pathways have been linked to asthma, inflammatory
arthritis,
atherosclerosis,
diabetic
vasculopathy,
osteoporosis, neuro-degenerative diseases, Down’s syndrome and immune system diseases. Abcam's Hydrogen Peroxide Assay Kit is a highly sensitive, simple, direct and HTS-ready colorimetric and fluorometric assay for measuring H2O2 in biological samples. In the presence of Horse Radish Peroxidase (HRP), the OxiRed Probe reacts with H2O2 to produce product with color (λmax = 570 nm) and red-fluorescent (Ex/Em=535/587 nm). The kit can perform 200 reactions by fluorometric method or 100 reactions by colorimetric method. The detection limit can be as low as 2 pmol per assay (or 40 nM concentration) of H2O2 in the sensitive fluorometric assay.
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2. Protocol Summary Sample Preparation
Standard Curve Preparation Prepare and Add Reaction Mix
Measure Optical Density or Fluorescence
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3. Components and Storage A. Kit Components Item
Quantity
H2O2 Assay Buffer
25 mL
OxiRed Probe Red
1 vial
Dimethylsulfoxide (DMSO, anhydrous) HRP H2O2 Standard (0.88M)
400 µL 1 vial 100 µL
* Store kit at -20°C, protect from light. Warm the Assay Buffer to room temperature before use. Briefly centrifuge all small vials prior to opening. Read the entire protocol before performing the assay. OxiRed PROBE: Dissolve in 220 µl DMSO (provided), pipetting up and down. The OxiRed Probe solution is stable for 1 week at 4°C and 1 month at -20°C. HRP: Dissolve in 220 µl assay buffer, pipetting up and down. The HRP solution is stable for 1 week at 4°C and 1 month at -20°C.
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B. Additional Materials Required •
Microcentrifuge
•
Pipettes and pipette tips
•
Fluorescent or colorimetric microplate reader
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96 well plate
•
Orbital shaker
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4. Assay Protocol 1. Sample Preparation: a) Collect cell culture supernatant, serum, plasma, urine and other biological fluids (contains 0.8-6 µM H2O2). b) Centrifuge for 15 minutes at 1000 x g within 30 minutes of collection. Remove particulate pellet. c) Assay immediately or aliquot and store the samples at -80°C. Avoid repeated freeze-thaw cycles. d) Add 2-50 µl samples into each well; bring the volume to 50 µl with assay buffer. 2. Standard Curve Preparation: a. For the colorimetric assay: Dilute 10 µl 0.88M H2O2 standard into 870 µl dH2O to generate 10 mM H2O2 standard, then dilute 10 µl 10 mM H2O2 standard into 990 µl dH2O to generate 0.1 mM H2O2 standard. Add 0, 10, 20, 30, 40, 50 µl of the 0.1 mM H2O2 standard into 96-well plate in duplicate to generate 0, 1, 2, 3, 4, 5 nmol/well H2O2 standard.
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b. For the fluorometric assay: Dilute 100 µl of the 0.1 mM H2O2 standard into 900 µl dH2O to generate 10 µM H2O2 Standard. Add 0, 10, 20, 30, 40, 50 µl of the 10 µM H2O2 standard into 96well plate in duplicate to generate 0, 0.1, 0.2, 0.3, 0.4, 0.5 nmol/well H2O2 standard. 3. Reaction Mix: Mix enough reagents for the number of assays to be performed. For each well, prepare a total 50 µl Reaction Mix: Colorimetric Assay
Fluorometric Assay
Assay Buffer
46 µl
48 µl
OxiRed Probe
2 µl
1 µl
HRP
2 µl
1 µl
Add 50 µl of the Reaction Mix to each test sample and H2O2 standards. Mix well. Incubate at room temperature for 10 min. Note: For a more sensitive assay, you can dilute the standard 10 fold further by decreasing OxiRed amount to 0.2 µl and HRP amount to 0.4 µl per well. This will decrease the fluorescence background, detecting as low as 2 pmol/well (or 40 µM concentration) H2O2. 4. Measure: Read OD570nm or fluorescence (Ex/Em = 535/587 nm) in a micro-plate reader.
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5. Data Analysis Correct background by subtracting the value derived from the zero nmol H2O2 control from all sample and standard readings. The background reading can be significant and must be subtracted from sample readings. Plot the H2O2 standard curve. Apply your sample readings to the standard curve. H2O2 concentrations of the test samples can then be calculated: Concentration = Sa / Sv (pmol/µl or µM) Where: Sa is the sample amount from your standard curve (in pmol), Sv is sample volume (µl).
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H2O2 Standard Curves performed according to Assay Protocol.
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6. Troubleshooting Problem
Reason
Solution
Assay not working
Assay buffer at wrong temperature
Assay buffer must not be chilled - needs to be at RT
Protocol step missed Plate read at incorrect wavelength
Unsuitable microtiter plate for assay
Unexpected results
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
Measured at wrong wavelength
Use appropriate reader and filter settings described in datasheet
Samples contain impeding substances Unsuitable sample type Sample readings are outside linear range
Troubleshoot and also consider deproteinizing samples Use recommended samples types as listed on the datasheet Concentrate/ dilute samples to be in linear range
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Samples with inconsistent readings
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
Lower/ Higher readings in samples and standards
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)
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Problem
Reason
Solution
Standard curve is not linear
Not fully thawed kit components
Wait for components to thaw completely and gently mix prior use
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
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 (
[email protected]) or phone (select “contact us” on www.abcam.com for the phone number for your region).
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