Description
Malate Assay Kit (BA0129) (BA0129)
This Malate Assay Kit (SKU: BA0129) provides a fast and sensitive colorimetric method for the quantitative determination of L-malate (L-malic acid) in food, juice, beverage and other agricultural products. L-malic acid is a dicarboxylic acid made by all living organisms that plays an important role in the Calvin and Krebs cycles and is frequently used as an additive in wine, beer and confectionery. The assay is based on malate dehydrogenase catalysed oxidation of malate, in which the NADH formed reduces a formazan (MTT) reagent. The intensity of the product colour, measured at 565 nm, is proportional to the malate concentration in the sample. The procedure uses only 20 µL of sample, a single working reagent and a 15-minute room-temperature incubation, and is readily automated for high-throughput use.
| Product Name: | Malate Assay Kit (BA0129) |
| SKU: | BA0129 |
| Detection Method: | Colorimetric (565 nm; range 520-600 nm) |
| Detection Range: | 0.02 to 2 mM L-malate (96-well plate assay) |
| Sample Type: | Food, juice, beverage and other agricultural products |
| Species Reactivity: | All |
| Assay Time: | 15 minutes |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | Store all components at -20°C upon receipt. |
| Shelf Life: | 6 months after receipt. |
| Shipping: | Gel Pack |
Malate dehydrogenase catalyses the oxidation of L-malate, and the NADH formed reduces a formazan (MTT) reagent. The intensity of the product colour, measured at 565 nm, is proportional to the L-malate concentration in the sample.
- Fast and sensitive, using 20 µL of sample with a linear detection range of 0.02 to 2 mM L-malate in a 96-well plate assay.
- Convenient: the procedure involves adding a single working reagent and reading the optical density at 15 minutes at room temperature, with no 37°C heater required.
- High-throughput and readily automated for thousands of samples per day.
- Direct assay of malate in food, juice, beverage and other agricultural products.
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: clear and slightly coloured samples may be assayed directly; homogenise solid samples (food, fruits, etc.) in water followed by filtration or centrifugation (e.g. 5 min at 14,000 rpm). |
| 2 | Beverage samples may be assayed directly; check the pH and adjust to 7-8 with NaOH or HCl if necessary, and degas carbonated samples by gentle stirring; test several dilutions to determine an optimal dilution factor n and store samples at -20 to -80°C for at least one month. |
| 3 | Equilibrate all components to room temperature and prepare 500 µL of 2.0 mM L-malate Premix by mixing 50 µL of the 20 mM Standard with 450 µL distilled water, then dilute the standards as described in the standard table. |
| 4 | Transfer 20 µL of each standard into separate wells of a clear flat-bottom 96-well plate and transfer 20 µL of each sample into two wells (one sample blank well and one sample well). |
| 5 | Prepare sufficient Working Reagent by mixing, per standard and sample well, 74 µL Assay Buffer, 1 µL Enzyme A, 1 µL Enzyme B and 8 µL NAD/MTT; prepare blank Working Reagent (per sample blank well) with 74 µL Assay Buffer, 1 µL Enzyme B and 8 µL NAD/MTT (no Enzyme A), and reconstitute the reagents freshly. |
| 6 | Add 80 µL Working Reagent to the four standards and the sample wells and 80 µL blank Working Reagent to the sample blank wells, tap to mix briefly and thoroughly and incubate 15 minutes at room temperature. |
| 7 | Read the optical density at 565 nm (520-600 nm). |
Subtract the blank value (standard #4) from the standard values and plot the change in OD against standard concentrations to determine the slope. [L-Malate] = (ODSAMPLE - ODBLANK) / Slope x n (mM), where ODSAMPLE and ODBLANK are the optical density readings of the sample and sample blank and n is the dilution factor. If the sample OD is higher than the OD for the 2 mM standard, dilute in water and repeat, multiplying the result by the dilution factor. Conversions: 1 mM L-malate equals 13.3 mg/dL, 0.018% or 133 ppm.
| Component | Quantity | Storage |
| Assay Buffer | 10 mL | -20°C |
| Enzyme A | 120 µL | -20°C |
| NAD/MTT | 1 mL | -20°C |
| Enzyme B | 120 µL | -20°C |
| Standard (20 mM L-Malate) | 1.0 mL | -20°C |
