Hydrogen Peroxide Fluorescent Detection Kit
(3 references)-
- 抗原
- Hydrogen Peroxide
- 检测方法
- Fluorometric
- 应用范围
- Detection (D)
- 原理
- The DetectX® Hydrogen Peroxide Fluorescent Detection Kit is designed to quantitatively measure H2O2 in a variety of samples.
- 品牌
- DetectX®
- 样品类型
- Biological Buffers, Cell Culture Supernatant, Urine
- 组件
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Black 96 well Half Area Plates 2 Plates Corning Costar Plate 3694.
Hydrogen Peroxide Standard 220 μL Hydrogen Peroxide at 100 μM in a special stabilizing solution.
Assay Buffer Concentrate 25 mL A 5X buffer concentrate containing detergents and stabilizers.
Fluorescent Detection Reagent 5 mL A solution of the substrate in a special stabilizing buffer.
Horseradish Peroxidase Concentrate 60 μL A 100X concentrated solution of HRP in a special stabilizing solution. - 试剂未包括
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Repeater pipet with disposable tips capable of dispensing 25 μL. 96 well plate reader capable of reading fluorescence at 580-590 nm with excitation at 570-580 nm.
Set plate parameters for a 96-well Corning Costar 3694 plate.
See: http://www.ArborAssays. com/resources/lit.asp for plate dimension data.
Software for converting fluorescent intensity readings from the plate reader and carrying out four parameter logistic curve (4PLC) fitting.
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Hydrogen Peroxide Colorimetric Detection Kit
D Colorimetric Biological Buffers, Cell Culture Supernatant, Urine
SensoLyte® ADHP Hydrogen Peroxide Assay KitEAA Fluorometric
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- 应用备注
- Optimal working dilution should be determined by the investigator.
- 实验流程
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A hydrogen peroxide standard is provided to generate a standard curve for the assay and all sam- ples should be read off the standard curve.
Samples are mixed with the Fluorescent Substrate and the reaction initiated by addition of horseradish peroxidase.
The reaction is incubated at room temperature for 15 minutes.
The HRP reacts with the substrate in the presence of hydrogen peroxide to convert the colorless substrate into a fluorescent product.
The fluorescent product is read at 590 nm with excitation at 570 nm.
Increasing levels of H2O2 cause a linear increase in fluorescent product. - 试剂准备
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Assay Buffer Preparation Dilute Assay Buffer Concentrate 1:5 by adding one part of the concentrate to four parts of deion- ized water.
Once diluted this is stable at 4 °C for 3 months.
Horseradish Peroxidase (HRP) Preparation Dilute the HRP Stock solution 1:100 with Assay Buffer using the table below: HRP Dilution Table 1/2 Plate One Plate Two Plates HRP Stock 15 μL 30 μL 55 μL Assay Buffer 1.485 mL 2.97 mL 5.445 mL Total Volume 1.5 mL 3 mL 5.5 mL - 样品制备
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Dilute samples ≥ 1:10 with Assay Buffer prior to running in the assay.
- 实验流程
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Use the plate layout sheet on the back page to aid in proper sample and standard identification. Set plate parameters for a 96-well Corning Costar 3694 plate. .
1. Pipet 50 μL of samples or appropriate standards into duplicate wells in the plate.
2. Pipet 50 μL of Assay Buffer into duplicate wells as the Zero standard.
3. Add 25 μL of Fluorescent Substrate to each well using a repeater pipet.
4. Initiate the reaction by adding 25 μL of the HRP Preparation to each well using a repeater pipet.
5. Incubate at room temperature for 15 minutes.
6. Read the fluorescent emission at 585 ± 5 nm with excitation at 575 ± 5 nm. Please contact your plate reader manufacturer for suitable filter sets. - 结果分析
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Average the duplicate FLU readings for each standard and sample.
Create a standard curve by reducing the data using computer software capable of generating a four-parameter logistic curve (4PLC) fit, after subtracting the mean FLUs for the Zero wells.
The sample concentrations ob- tained should be multiplied by the dilution factor to obtain neat sample values.
Or use the online tool from www.myassays.com/arbor-assays-hydrogen-peroxide-fluorescent- detection-kit.assay to calculate the data. *The MyAssays logo is a registered trademark of MyAssays Ltd. typical data Sample Mean FLU Net FLU H2O2 Conc. (μM) Zero 3,782 0 0 Standard 1 36,417 32,635 10 Standard 2 21,919 18,137 5 Standard 3 13,134 9,352 2.5 Standard 4 8,333 4,551 1.25 Standard 5 6,072 2,290 0.625 Standard 6 5,031 1,249 0.313 Standard 7 4,398 616 0.156 Sample 1 6,578 2,796 0.76 Sample 2 24,680 20,898 5.85 Always run your own standard curves for calculation of results.
Do not use these data.
Conversion Factor: 100 nM of Hydrogen Peroxide is equivalent to 3.4 ng/mL. - 实验精密度
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Three buffer samples were run in replicates of 20 in an assay.
Inter Assay Precision:
Three buffer samples were run in duplicates in fourteen assays run over multiple days by three operators. - 限制
- 仅限研究用
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- 注意事项
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As with all such products, this kit should only be used by qualified personnel who have had labo- ratory safety instruction.
The supplied hydrogen peroxide standard contains very dilute H2O2.
- 注意事项
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Sample typeS and preparatiOn Samples that need to be stored after collection should be stored at -70°C or lower, preferably after being frozen in liquid nitrogen.
Urine samples can be used after being diluted ≥ 1:10.
This assay has been validated for buffer and media samples. - 储存条件
- 4 °C,RT
- 储存方法
- All components of this kit should be stored at 4°C until the expiration date of the kit.
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: "Reactive oxygen species induced by cold stratification promote germination of Hedysarum scoparium seeds." in: Plant physiology and biochemistry : PPB, Vol. 109, pp. 406-415, (2016) (PubMed).
: "Indirect effects of TiO2 nanoparticle on neuron-glial cell interactions." in: Chemico-biological interactions, Vol. 254, pp. 34-44, (2016) (PubMed).
: "Modification of PDMS to fabricate PLGA microparticles by a double emulsion method in a single microfluidic device." in: Lab on a chip, Vol. 16, Issue 14, pp. 2596-600, (2016) (PubMed).
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: "Reactive oxygen species induced by cold stratification promote germination of Hedysarum scoparium seeds." in: Plant physiology and biochemistry : PPB, Vol. 109, pp. 406-415, (2016) (PubMed).
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- 抗原
- Hydrogen Peroxide
- 物质类
- Anorganic
- 背景
- Hydrogen peroxide was first described in 1818 by Louis Jacques Thénard. Today, industrially, hydrogen peroxide is manufactured almost exclusively by the autoxidation of a 2-alkyl-9,10-dihy- droxyanthracene to the corresponding 2-alkyl anthraquinone in the Riedl-Pfleiderer or anthraqui- none process. In biological systems incomplete reduction of O2 during respiration produces superoxide anion (O -2 •), which is spontaneously or enzymatically dismutated by superoxide dismutase to H2O2. Many cells produce low levels of O -2 • and H2O2 in response to a variety of extracellular stimuli, such as cytokines (TGF-ß1, TNF-a, and various interleukins), peptide growth factors (PDGF, EGF, VEGF, bFGF, and insulin), the agonists of heterotrimeric G protein-coupled receptors (GPCR) such as an- giotensin II, thrombin, lysophosphatidic acid, sphingosine 1-phosphate, histamine, and bradykinin, and by shear stress1. The addition of exogenous H2O2 or the intracellular production in response to receptor stimulation affects the function of various proteins, including protein kinases, protein phosphatases, transcription factors, phospholipases, ion channels, and G proteins. In 1894, Fen- ton2 described the oxidation of tartaric acid by Fe2+ and H2O2. H2O2 and O2 may participate in the production of singlet oxygen and peroxynitrite and the generation of these species may be concurrent with reactions involving iron, and under some circumstances they might be important contributors to H O toxicity3,42 2 . A substantial portion of H2O2 lethality involves DNA damage by oxidants generated from iron- mediated Fenton reactions5,6. Damage by Fenton oxidants occurs at the DNA bases or at the sugar residues. Sugar damage is initiated by hydrogen abstraction from one of the deoxyribose carbons, and the predominant consequence is eventual strand breakage and base release7,8
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