Description
Xanthine Oxidase Assay Kit (BA0155) (BA0155)
The Xanthine Oxidase Assay Kit (SKU: BA0155) provides a simple, direct and high-throughput method for measuring xanthine oxidase activity in biological samples. Xanthine oxidase catalyses the oxidation of xanthine to uric acid and of hypoxanthine to xanthine, and its activity in blood can act as a marker for influenza, liver damage and cardiovascular health. This assay uses a single working reagent that combines the xanthine oxidase reaction and the colour reaction in one step. The change in colour intensity of the product at 570 nm, or the fluorescence intensity at 530/585 nm, is directly proportional to the xanthine oxidase activity in the sample. The assay uses as little as 10 uL of sample and is well suited to drug-screening applications.
| Product Name: | Xanthine Oxidase Assay Kit (BA0155) |
| SKU: | BA0155 |
| Detection Method: | Colorimetric / Fluorometric |
| Detection Range: | Colorimetric 0.03 - 25 U/L; fluorometric 0.01 - 2.5 U/L xanthine oxidase |
| Sample Type: | Cell lysate, serum and other biological samples |
| Species Reactivity: | All |
| Assay Time: | 20 minutes |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | Store all reagents at -20 C. |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
A single-reagent enzymatic assay for the quantitative colorimetric or fluorometric determination of xanthine oxidase activity. Xanthine oxidase generates hydrogen peroxide, which is measured through a coupled colour reaction at 570 nm or a fluorescence reaction at 530/585 nm, with signal proportional to enzyme activity. The procedure adds a single working reagent and reads over a 20 min room-temperature incubation.
- Sensitive and accurate, using as little as 10 uL of sample
- Linear detection range: colorimetric 0.03 - 25 U/L, fluorometric 0.01 - 2.5 U/L (20 min)
- Simple single-working-reagent format
- Suitable for high-throughput drug screening
- Direct measurement of xanthine oxidase activity in cell lysate, serum and other biological samples
- Studies of the effects of drugs on xanthine oxidase metabolism
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | Equilibrate all components to room temperature, keeping thawed enzyme refrigerated or on ice. Centrifuge samples and use the clear supernatant if particulates are present. |
| 2 | Prepare a hydrogen peroxide standard curve: dilute 3% H2O2 to 4.8 mM and then to 400 uM, and prepare standards at 400, 240, 120 and 0 uM. Transfer 10 uL of each standard and 10 uL of each sample into separate wells. |
| 3 | Prepare Working Reagent per well by mixing 85 uL Assay Buffer, 10 uL 5 mM Xanthine, 1 uL HRP Enzyme and 1 uL Dye Reagent. Transfer 90 uL to each well and tap to mix. |
| 4 | Read optical density immediately at 570 nm (OD0), incubate for 20 min at room temperature and read again (OD20). |
| 5 | For the fluorometric assay, dilute the standards to 40, 24, 12 and 0 uM, use a black plate and read fluorescence at 530/585 nm immediately (F0) and after 20 min (F20). |
Subtract the water blank from the standard OD20 or F20 values and plot against hydrogen peroxide concentration to determine the slope. For each sample calculate the change over 20 min (OD20 - OD0 or F20 - F0), then XO Activity = ((dR_SAMPLE - dR_BLANK) / (Slope x t)) x n (U/L), where t is 20 min and n is the dilution factor. One U/L of xanthine oxidase catalyses the conversion of 1 umole of xanthine to uric acid per minute at pH 7.0 and room temperature.
| Component | Quantity | Storage |
| Assay Buffer | 10 mL | -20 C |
| HRP Enzyme | 120 uL | -20 C |
| Xanthine (5 mM) | 1.5 mL | -20 C |
| Dye Reagent | 120 uL | -20 C |
| Standard (3% H2O2) | 100 uL | -20 C |
