Malate Assay Kit (Colorimetric)

Malate, the anion salt of malic-acid, is a dicarboxylic acid that is an important intermediate in various metabolic pathways which are central to cellular energy production. Malate is shuttled across the inner mitochondria by the malate-aspartate shuttle to transport reducing equivalents (NADH) into the mitochondrial matrix for ATP production. Additionally, malate is monitored during malolactic fermentation in the wine industry, alongside l-lactic acid. There has been considerable investigation of the benefits of malate to treat fibromyalgia, chronic fatigue syndrome, dry mouth, and as an agent in food and pharmaceuticals.

Malate Assay Kit provides a simple, reproducible, and sensitive tool of measuring malate concentration in plasma, serum, cell lysates, urine, and other biological liquid samples. This assay employs a coupled enzymatic reaction system that combines multiple steps. Initially, malate dehydrogenase catalyzes the conversion of malate to oxaloacetate. The generated NADH interacts with colorimetric probe to produce a yellow-orange colored compound that is measurable at 450nm. The optical density measured at 450nm is directly proportional to the concentration of malate present in the sample.

• Microplate reader capable of measuring absorbance at 450 nm
• Precision pipettes to deliver 2 µL to 1 mL volumes
• Tubes to prepare sample dilutions
• 15mL, 50mL conical tubes
• Orbital shaker capable of 37°C
• MilliQ water or another type of double distilled water (ddH₂O)

1. Add 40 µL of the diluted L-Malate Standards or samples to the 96-well microtiter plate.
2. Add 200 µL Reaction Mix to each standard and one half of the paired sample wells, mix thoroughly.
3. Add 200 µL of Control Mix to other half of the paired sample wells. Carefully shake the plate for a few seconds to mix.
4. Seal the plate with a plate sealer or plastic wrap to reduce evaporation. Incubate the plate for 60 minutes at 37°C with mixing in the dark.
5. Measure the absorbance at 450 nm using a plate reader.