Description
Xanthine Assay Kit (Colorimetric or Fluorometric) (BA0154) (BA0154)
The Xanthine Assay Kit (SKU: BA0154) provides a simple, direct and high-throughput method for measuring xanthine in biological samples. Xanthine is a purine base found in most animal tissues and fluids, produced in the purine degradation pathway and degraded to uric acid by xanthine oxidase, and its derivatives act as antagonists at sleep-inducing adenosine receptors. 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 concentration in the sample. The assay uses as little as 10 uL of sample and is well suited to high-throughput screening.
| Product Name: | Xanthine Assay Kit (Colorimetric or Fluorometric) (BA0154) |
| SKU: | BA0154 |
| Detection Method: | Colorimetric / Fluorometric |
| Detection Range: | Colorimetric 0.01 - 2 mM; fluorometric 3 - 250 uM xanthine |
| Sample Type: | Cell lysate, serum and other biological samples |
| Species Reactivity: | All |
| Assay Time: | 30 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. Xanthine oxidase and a coupled colour reaction yield a product measured at 570 nm or a fluorescent product at 530/585 nm, with signal proportional to xanthine concentration. The procedure adds a single working reagent and reads after a 30 min room-temperature incubation.
- Sensitive and accurate, using as little as 10 uL of sample
- Linear detection range: colorimetric 0.01 - 2 mM, fluorometric 3 - 250 uM xanthine (30 min)
- Simple single-working-reagent format
- Fast and high-throughput
- Direct measurement of xanthine in cell lysate, serum and other biological samples
- Studies of the effects of drugs on xanthine (purine) 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 standards at 2, 1.2, 0.6 and 0 mM. Transfer 10 uL of each standard and 10 uL of each sample into separate wells of a clear flat-bottom 96-well plate. |
| 3 | Prepare Working Reagent per well by mixing 90 uL Assay Buffer, 1 uL XO Enzyme, 1 uL HRP Enzyme and 1 uL Dye Reagent. Transfer 90 uL to each well and tap to mix. |
| 4 | Incubate for 30 min at room temperature and read optical density at 570 nm (550-585 nm). |
| 5 | For the fluorometric assay, dilute the standards to 200, 120, 60 and 0 uM, use a black plate and read fluorescence at 530/585 nm after 30 min. |
Subtract the water blank from all standard and sample values and plot the change in optical density or fluorescence against xanthine concentration to determine the slope. Calculate [Xanthine] = ((R_SAMPLE - R_BLANK) / Slope) x n, where R is the sample or blank reading and n is the dilution factor. If the concentration exceeds the linear range, dilute the sample in water and repeat.
| Component | Quantity | Storage |
| Assay Buffer | 10 mL | -20 C |
| HRP Enzyme | 120 uL | -20 C |
| Standard (2 mM Xanthine) | 1 mL | -20 C |
| Dye Reagent | 120 uL | -20 C |
| XO Enzyme | 100 uL | -20 C |
