Description
Pyruvate Assay Kit (Colorimetric or Fluorometric) (BA0146) (BA0146)
The Pyruvate Assay Kit (SKU: BA0146) provides a simple, direct and automation-ready procedure for measuring pyruvate concentration. Pyruvate is a key intermediate in cellular metabolism that can be converted to carbohydrates via gluconeogenesis, to fatty acids or energy through acetyl-CoA, to the amino acid alanine and to ethanol, and abnormal levels have been linked to liver diseases and metabolic disorders. This assay uses a single working reagent that combines pyruvate oxidase with hydrogen-peroxide determination in one step. The colour intensity of the product at 570 nm, or the fluorescence intensity at 585/530 nm, is directly proportional to the pyruvate concentration in the sample. The optimised formulation enhances reagent and signal stability and the assay uses as little as 10 uL of sample.
| Product Name: | Pyruvate Assay Kit (Colorimetric or Fluorometric) (BA0146) |
| SKU: | BA0146 |
| Detection Method: | Colorimetric / Fluorometric |
| Detection Range: | Colorimetric 2 - 500 uM; fluorometric 0.2 - 50 uM pyruvate |
| Sample Type: | 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 pyruvate. Pyruvate oxidase generates hydrogen peroxide, which reacts with a dye to yield a coloured product measured at 570 nm or a fluorescent product measured at 585/530 nm. The procedure involves a single 30 min incubation at room temperature and is compatible with high-throughput screening.
- Sensitive and accurate, using as little as 10 uL of sample
- Linear detection range in 96-well plate: colorimetric 2 - 500 uM, fluorometric 0.2 - 50 uM pyruvate
- Simple single-working-reagent format with a 30 min room-temperature incubation
- Improved reagent and signal stability
- Direct measurement of pyruvate in biological samples
- Studies of the effects of drugs on pyruvate 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. |
| 2 | Prepare standards: make a 500 uM premix (10 uL of 25 mM Standard in 490 uL water) and dilute to 500, 400, 300, 200, 150, 100, 50 and 0 uM. Transfer 10 uL of each standard and 10 uL of each sample into wells of a clear flat-bottom 96-well plate. |
| 3 | Prepare Working Reagent per well by mixing 94 uL Enzyme Mix 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 1:10, use a black plate and read fluorescence at 530/585 nm after 30 min. A 384-well format is also supported using 5 uL standards and 45 uL Working Reagent. |
Subtract the water blank from the standard values and plot the optical density or fluorescence against pyruvate concentration to determine the slope by linear regression. Calculate [Pyruvate] = (R_SAMPLE - R_WATER) / Slope (uM). If the reading exceeds the top standard, dilute the sample in water and repeat. One mM pyruvate equals 8.7 mg/dL or 87 ppm.
| Component | Quantity | Storage |
| Enzyme Mix | 10 mL | -20 C |
| Dye Reagent | 120 uL | -20 C |
| Standard (25 mM Pyruvate) | 400 uL | -20 C |
