For the rapid, sensitive and accurate measurement of Hydrogen Peroxide in various samples

Transcription

1 ab 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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3 Table of Contents 1. Overview 3 2. Protocol Summary 4 3. Components and Storage 5 4. Assay Protocol 7 5. Data Analysis 9 6. Troubleshooting 11 2

4 1. Overview Hydrogen Peroxide (H 2 O 2 ) 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 H 2 O 2 in biological samples. In the presence of Horse Radish Peroxidase (HRP), the OxiRed Probe reacts with H 2 O 2 to produce product with color (λ max = 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 H 2 O 2 in the sensitive fluorometric assay. 3

5 2. Protocol Summary Sample Preparation Standard Curve Preparation Prepare and Add Reaction Mix Measure Optical Density or Fluorescence 4

6 3. Components and Storage A. Kit Components Item Quantity H 2 O 2 Assay Buffer 25 ml OxiRed Probe Red 200 µl HRP 1 vial H 2 O 2 Standard (0.88M) 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 Red: Ready to use as supplied. Briefly warm at 37 C to melt frozen DMSO. TheOxiRed 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. 5

7 B. Additional Materials Required Microcentrifuge Pipettes and pipette tips Fluorescent or colorimetric microplate reader 96 well plate Orbital shaker 6

8 4. Assay Protocol 1. Sample Preparation: a) Collect cell culture supernatant, serum, plasma, urine and other biological fluids (contains μm H 2 O 2 ). b) Centrifuge for 15 minutes at 1000 x g within 30 minutes of collection. Remove particulate pellet. c) Samples, especially those such as culture medium, tissue lysate or plasma should be filtered through a 10 kda MW spin filter to remove all proteins then kept at -80 C for storage. d) It is recommended with all sample types to assay immediately or aliquot and store the samples at -80 C. Avoid repeated freezethaw cycles e) Add 2-50 μl samples into each well; bring the volume to 50 μl with assay buffer. 7

9 2. Standard Curve Preparation: a. For the colorimetric assay: Dilute 10 μl 0.88M H 2 O 2 standard into 870 μl dh 2 O to generate 10 mm H 2 O 2 standard, then dilute 10 μl 10 mm H 2 O 2 standard into 990 μl dh 2 O to generate 0.1 mm H 2 O 2 standard. Add 0, 10, 20, 30, 40, 50 μl of the 0.1 mm H 2 O 2 standard into 96-well plate in duplicate to generate 0, 1, 2, 3, 4, 5 nmol/well H 2 O 2 standard. b. For the fluorometric assay: Dilute 100 μl of the 0.1 mm H 2 O 2 standard into 900 μl dh 2 O to generate 10 μm H 2 O 2 Standard. Add 0, 10, 20, 30, 40, 50 μl of the 10 μm H 2 O 2 standard into 96- well plate in duplicate to generate 0, 0.1, 0.2, 0.3, 0.4, 0.5 nmol/well H 2 O 2 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 H 2 O 2 standards. Mix well. Incubate at room temperature for 10 min. 8

10 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) H 2 O Measure: Read OD 570nm or fluorescence (Ex/Em = 535/587 nm) in a micro-plate reader. 5. Data Analysis Correct background by subtracting the value derived from the zero nmol H 2 O 2 control from all sample and standard readings. The background reading can be significant and must be subtracted from sample readings. Plot the H 2 O 2 standard curve. Apply your sample readings to the standard curve. H 2 O 2 concentrations of the test samples can then be calculated: Where: Concentration = Sa / Sv (pmol/μl or μm) Sa is the sample amount from your standard curve (in pmol), Sv is sample volume (μl). 9

11 H 2 O 2 Standard Curves performed according to Assay Protocol. 10

12 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

13 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

14 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 (technical@abcam.com) or phone (select contact us on for the phone number for your region). 13

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16 UK, EU and ROW Tel: +44 (0) US, Canada and Latin America Tel: ABCAM (22226) China and Asia Pacific Tel: ( 中國聯通 ) Japan technical@abcam.co.jp Tel: +81-(0) Copyright 2012 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. 15 All information / detail is correct at time of going to print.