Description
Fumarate Assay Kit (BA0110) (BA0110)
The Fumarate Assay Kit (SKU: BA0110) offers a fast, sensitive colorimetric method for determining fumarate (fumaric acid). Fumarate is a key component of the TCA cycle used by cells to form ATP, and is widely used as a food and beverage additive; elevated urinary fumarate may indicate impaired Krebs cycle function or a fumarase defect. The assay is based on fumarase-catalysed hydration of fumarate to malate, which is then oxidised by malate dehydrogenase to generate NADH that reduces an MTT dye. The intensity of the product colour, measured at 565 nm, is proportional to the fumarate concentration. The room-temperature procedure requires no 37C heater and is readily automated.
| Product Name: | Fumarate Assay Kit (BA0110) |
| SKU: | BA0110 |
| Detection Method: | Colorimetric (565 nm) |
| Detection Range: | 0.005 - 2 mM fumarate |
| Sample Type: | Food, beverage, cell lysate, tissue homogenate and serum |
| Species Reactivity: | All |
| Assay Time: | 30 minutes |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20C |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
A colorimetric assay for fumarate in food, beverage and biological samples using only a 20 uL sample. Samples containing malate require a sample blank.
- Fast and sensitive using only a 20 uL sample, with a linear range of 0.005 - 2 mM fumarate
- Convenient single working reagent read after 30 minutes at room temperature, no 37C heater required
- High-throughput and readily automated
- Direct assay of fumarate in food, beverage and other biological samples such as cell lysate, tissue homogenate and serum
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 | Sample preparation: homogenise solid samples in water and filter or centrifuge; beverage samples can be assayed directly. Adjust pH to 7-8 if necessary and degas carbonated samples. Homogenise tissue in 50 mM potassium phosphate (pH 7.5) and centrifuge at 10,000 g for 15 min at 4C. |
| 2 | Standards: prepare 200 uL of 2 mM Premix by mixing 20 uL of the 20 mM Standard with 180 uL distilled water, then dilute per the table. Transfer 20 uL of each standard into a clear flat-bottom 96-well plate. |
| 3 | Transfer 20 uL of each sample into separate wells. If a sample is known to contain malate, add it to two wells (one serving as a sample blank). |
| 4 | Prepare Working Reagent per well from 74 uL Assay Buffer, 8 uL NAD/MTT, 1 uL Enzyme A, 1 uL Enzyme B and 1 uL FMR Enzyme. For sample blanks, omit the FMR Enzyme (use 75 uL Assay Buffer). |
| 5 | Add 80 uL of the appropriate reagent per well and tap to mix. |
| 6 | Incubate at room temperature for 30 minutes and read OD565nm (520-600 nm). |
Subtract the blank value from the standard values and plot delta-OD against standard concentration. [Fumarate] = (ODSample - ODBlank) / Slope x n (mM), where n is the sample dilution factor. If a reading exceeds that of the 2 mM standard, dilute in water, repeat and multiply by the dilution factor. 1 mM fumarate equals 11.6 mg/dL or 116 ppm.
| Component | Quantity | Storage |
| Assay Buffer | 10 mL | -20C |
| NAD/MTT | 1 mL | -20C |
| Standard (20 mM Fumarate) | 1 mL | -20C |
| Enzyme A | 120 uL | -20C |
| Enzyme B | 120 uL | -20C |
| FMR Enzyme | 120 uL | -20C |
