OxiSelect™ Hydrogen Peroxide/Peroxidase Assay Kit (Colorimetric)

Produktnummer ABIN5067620

Produktdetails

Reaktivität: Others

Nachweismethode: Colorimetric

Applikation: Biochemical Assay (BCA)

Verwendungszweck: The OxiSelect™ Hydrogen Peroxide/Peroxidase Assay Kit is a sensitive quantitative colorimetric assay for hydrogen peroxide or peroxidase.

Marke: OxiSelect™

Analytische Methode: Quantitative

Sensitivität: 500 nM,0.16 mU/mL

Produktmerkmale: OxiSelect™ Hydrogen Peroxide/Peroxidase Assay Kit is a simple HTS-compatible assay for measuring hydrogen peroxide concentrations or peroxidase activities in biological samples without any need for pretreatment. The colorimetric probe reacts with H2O2 and horseradish peroxidase enzyme (HRP) to produce a pink colored product. The probe has less background and greater stability than the commonly used Xylenol Orange (FOX) colorimetric assay for H2O2. The probe can be also used as an ultrasensitive assay for peroxidase activity when H2O2 is in excess. The kit has a detection sensitivity limit of 500 nM (H2O2) or 0.16 mU/mL (peroxidase). Each kit provides sufficient reagents to perform up to 500 assays, including standard curve and unknown samples.

Bestandteile:

1. Colorimetric Probe (100X) : One 250 μL amber tube of solution.
2. HRP : One 100 μL tube of a 100 U/mL solution in glycerol*.
3. Hydrogen Peroxide : One 100 μL amber tube of an 8.8 M solution.
4. 10X Assay Buffer : One 25 mL bottle. *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.

Anwendungsinformationen

Applikationshinweise: Optimal working dilution should be determined by the investigator.

Protokoll: In the presence of peroxidase, the probe reacts with H2O2 in a 1:1 stoichiometry to produce a bright pink colored product. This product can be easily read by a standard colorimetric microplate reader with a filter in the 540-570 nm range. Absorbance values are proportional to the H2O2 or peroxidase levels within the samples, depending on the assay employed. The H2O2 or peroxidase content in unknown samples is determined by comparison with its respective standard curve.

Aufbereitung der Reagenzien:

Note: All reagents must be brought to room temperature prior to use. 3

• 1X Assay Buffer: Dilute the stock 10X Assay Buffer 1:10 with deionized water for a 1X solution. Stir or vortex to homogeneity.
• Hydrogen Peroxide Working Solution (Hydrogen Peroxide Assay): If measuring hydrogen peroxide, prepare a working solution by diluting the Colorimetric Probe 1:100 and HRP to a final concentration of 0.2 U/mL in 1X Assay Buffer (eg. Add 50 μL Colorimetric Probe stock solution and 10 μL HRP stock solution to 4.940 mL 1X Assay Buffer). This volume is enough for ~100 assays. The Hydrogen Peroxide Working Solution should be protected from light and used within 4 hours. Prepare only enough for immediate use.
• Peroxidase Working Solution (Peroxidase Assay): If measuring peroxidases, prepare a working solution by diluting the Colorimetric Probe 1:100 and H2O2 to a final concentration of 2 mM in 1X Assay Buffer. First perform a 1:1000 dilution of the stock H2O2 in 1X Assay Buffer. Use only enough for immediate applications (eg. Add 5 μL of H2O2 to 4.995 mL 1X Assay Buffer). This solution has a concentration of 8.8 mM. Use this 8.8 mM H2O2 solution to prepare a 2 mM H2O2 solution in Probe/1X Assay Buffer (eg. Add 50 μL Colorimetric Probe stock solution and 1.14 mL of the prepared 8.8 mM H2O2 solution to 3.81 mL 1X Assay Buffer). This volume is enough for ~100 assays. The Peroxidase Working Solution should be protected from light and used within 4 hours. Prepare only enough for immediate use.

Aufbereitung der Proben:

• 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 H2O2 standard curve in the same non-conditioned media. Serum should be avoided, as it interferes with the assay. Note: Maintain pH between 7 and 8 for optimal working conditions as the probe is unstable at high pH (>8.5).
• Cell lysate: Resuspend cells at 1-2 x 106 cells/mL in PBS or 1X Assay Buffer. Homogenize or sonicate the cells on ice. Centrifuge to remove debris. Cell lysates can be assayed undiluted or titrated as necessary.
• Plasma or urine: To remove insoluble particles, centrifuge at 10,000 rpm for 5 min. The supernatant can be assayed directly or diluted as necessary. Notes:
• All samples 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.
• A serial dilution will be necessary depending on the total H2O2 or peroxidase present. Extremely high levels of H2O2 (≥ 500 μM final concentration) or peroxidase (≥ 100 mU/mL) can lower the absorbance because excess H2O2 or peroxidase can further oxidize the reaction product.
• Samples with NADH concentrations above 10 μM and glutathione concentrations above 50 μM will oxidize the 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 (Tatyana et al, Ref. 2).
• Avoid samples containing DTT or β-mercaptoethanol since the probe is not stable in the presence of thiols (above 10 μM). 4

Testdurchführung:

I. Hydrogen Peroxide

1. Prepare and mix all reagents thoroughly before use. Each sample, including unknowns and standards, should be assayed in duplicate or triplicate.
2. Add 50 μL of each sample (H2O2 standard, control or unknown) into an individual microtiter plate well.
3. Add 50 μL of Hydrogen Peroxide Working Solution to each well. Mix the well contents thoroughly and incubate for 30 minutes at room temperature 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.
4. Read the plate absorbance with a microplate reader in the 540-570 nm range.
5. Calculate the concentration of peroxide within samples by comparing the sample absorbance to the standard curve. Subtract the value from the zero H2O2 control.

II. Peroxidase

1. Prepare and mix all reagents thoroughly before use. Each sample, including unknowns and standards, should be assayed in duplicate or triplicate.
2. Add 50 μL of each sample (HRP standard, control or unknown) into an individual microtiter plate well.
3. Add 50 μL of Peroxidase Working Solution to each well. Mix the well contents thoroughly and incubate for 30 minutes at room temperature 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.
4. Read the plate absorbance with a microplate reader in the 540-570 nm range.
5. Calculate the concentration of peroxidase within samples by comparing the sample absorbance to the standard curve. Subtract the value from the zero HRP control.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Handhabung: Avoid multiple freeze/thaw cycles.

Lagerung: 4 °C/-20 °C

Informationen zur Lagerung: Upon receipt, aliquot and store the Colorimetric Probe and HRP at -20°C. Avoid multiple freeze/thaw cycles. Store the remaining kit components at 4°C. The Colorimetric Probe is light sensitive and must be stored accordingly.

Antigendetails

Hintergrund: Oxidative stress is a physiological condition where there is an imbalance between concentrations of reactive oxygen species (ROS) and antioxidants. Research has shown that excessive ROS accumulation will lead to cellular injury, such as damage to DNA, proteins, and lipid membranes. Peroxides, such as hydrogen peroxide (H2O2), are some of the most well documented ROS produced under oxidative stress conditions. 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 peroxides have been implicated in the development of many pathological conditions, such as ageing, asthma, arthritis, diabetes, cardiovascular disease, atherosclerosis, Down's Syndrome, and neurodegenerative diseases.