Peroxide Assay Kit, BioAssay(TM), Colorimetric

Product Overview

• Article Name: Peroxide Assay Kit, BioAssay(TM), Colorimetric
• Biozol Catalog Number: USB-P3351-71
• Supplier Catalog Number: P3351-71
• Alternative Catalog Number: USB-P3351-71-250
• Manufacturer: US Biological
• Category: Kits/Assays

Product Description

Peroxide (e.g. hydrogen peroxide H2O2) is one of the key reactive oxygen species formed under oxidative stress conditions. High levels of peroxide formation have been linked to pathological conditions such as ageing, asthma, diabetes, atherosclerosis, cataract, inflammatory arthritis and neurodegenerative diseases. Simple, direct and automation-ready procedures for quantitative determination of peroxide find wide applications in research and drug discovery. Peroxide assay kit is designed to measure peroxide concentration in biological samples without any pretreatment. The improved method utilizes the chromogenic Fe3+-xylenol orange reaction, in which a purple complex is formed when Fe2+ provided in the reagent is oxidized to Fe3+ by peroxides present in the sample. The intensity of the color, measured at 540-610nm, is an accurate measure of the peroxide level in the sample. The optimized formulation substantially reduces interference by substances in the raw samples. Key Features: Sensitive and accurate. Enhanced color intensity using sorbitol. Detection range 0.2uM (7 ng/mL) to 30uM (1,020 ng/mL) H2O2 in 96-well plate assay. Simple and high-throughput. The procedure involves addition of a single detection reagent and incubation for 30 min. Can be readily automated as a high-throughput assay in 96-well plates for thousands of samples per day. Applications: Direct Assays: H2O2 in biological samples (e.g. serum, citrate-plasma, urine, cell lysate, culture medium). Pharmacology: effects of drugs on peroxide metabolism. Kit Contents: (250 tests in 96-well plates) Reagent A: 1ml Reagent B: 50ml Standard: 100ul 3% stabilized H2O2. Kit shipped at room temperature. Storage conditions. The kit is shipped at room temperature. Store all reagents at 4 C. Shelf life: 6 months after receipt. Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information. Procedures: Sample Treatment: several chemicals are known to interfere and should be avoided in sample preparation. These include ascorbic acid, EDTA, heparin, DMSO (>0.02%), NP-40 (>0.6%), SDS (>0.12%), Tris (>8mM) and ethanol (>0.4%). Samples can be analyzed immediately after collection, or stored in aliquots at -20 C. Avoid repeated freeze-thaw cycles. Reagent Preparation: Equilibrate to room temperature before assay. Prepare enough Detection Reagent by mixing 1 volume of Reagent A with 100 volumes Reagent B. Procedure using 96-well plate: 1. Standards. Prepare fresh standards on the day of assay. Pipette 5ul 3% H2O2 and mix well with 495ul H2O in a 1.5-mL Eppendorf tube. Mix 5ul of this solution with 1465ul H2O. The final H2O2 concentration is 30uM (labeled ''Premix). Dilute standard as shown in the Table. 2. Transfer 40ul diluted standards and each sample into separate wells of a clear flat-bottom 96-well plate. Add 200ul Detection Reagent to all standards and samples. No Premix+H2O Vol (ul) H2O2 (uM) 1 100ul+0ul 100 30 2 80ul+20ul 100 24 3 60ul+40ul 100 18 4 40ul+60ul 100 12 5 30ul+70ul 100 9 6 20ul+80ul 100 6 7 10ul+90ul 100 3 8 0ul+100ul 100 0 3. Incubate 30 min at room temperature and read optical density at 540-610nm (peak absorbance at 585nm). Note: if in rare cases, precipitation occurs after adding the Detection Reagent to a sample, transfer the whole reaction mixture of this sample well into a 1.5-mL Eppendorf tube and centrifuge 2 min at 14,000 rpm. Carefully remove 200ul supernatant into a clean well and read OD. Multiply the OD reading by 1.2 to account for the volume change. Calculation: Subtract blank OD (water, 8) from the standard OD values and plot the OD against H2O2 concentrations. Subtract blank OD from Sample OD. Determine the sample peroxide content from the standard curve. Conversions: 1uM H2O2 equals 34 ng/mL or 34 ppb. Materials Required, But Not Provided: Pipetting devices and accessories, 96-well plates and plate r