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Cat. No.: LMAK-100 (for 100T)

Description

L-Malate Assay Kit is a high-sensitivity colorimetric assay kit based on the WST-8 reaction. It enables rapid and sensitive detection of L-malic acid and its salts in biological fluids such as tissue or cell samples, serum, and plasma, as well as in beverages and juices.

Malic acid (Malate), also known as 2-hydroxybutanedioic acid, has the molecular formula C₄H₆O₅ and a molecular weight of 134.09. Malic acid is a dicarboxylic acid produced by organisms and is an essential organic acid for humans. There are two isomers of malic acid: L-malic acid and D-malic acid. The naturally occurring form is almost exclusively L-malic acid. L-malic acid is an important component of natural fruit juices, providing a taste similar to that of natural apples. Compared to citric acid, it offers a better taste with lower caloric content and is considered the "ideal food acidulant." It is one of the most widely used and promising organic acids in the global food industry. Malic acid is a crucial intermediate in the body's internal cycle and is easily absorbed, making it a popular additive in food, cosmetics, medical, and health products. It is the third most used food acidulant after citric acid and lactic acid, and its quantitative measurement is important for understanding related conditions and food production.

As an important intermediate in the tricarboxylic acid cycle (TCA cycle), malic acid plays a vital role in metabolism. In the C4 fixation process of plants, malic acid is a source of CO₂ in the Calvin cycle. In lower organisms, malic acid is converted to lactic acid through malic acid-lactic acid fermentation by forming CO₂. Malic acid can also be synthesized through the carboxylation of phosphoenolpyruvate in plant guard cells. As a dicarboxylate, malic acid often accompanies potassium cations during solute uptake into guard cells to maintain intracellular electrical balance. The accumulation of these solutes in guard cells lowers the solute potential, allowing water to enter the cells and promoting the opening of stomata.

Components

• Buffer A for Metabolic Assay
• Malic Acid Detection Buffer
• Enzyme Solution
• Color Development Solution
• Substrate
• Malic Acid Standard Solution (10 mM)

Principle

L-malic acid is oxidized to oxaloacetic acid (OAA) under the action of malate dehydrogenase (MDH), during which NAD⁺ is reduced to NADH. The generated NADH then reduces WST-8 to produce an orange-yellow formazan in the presence of the electron coupling reagent 1-mPMS (1-Methoxy-5-methylphenazinium methyl sulfate), with a maximum absorbance peak around 450 nm. The amount of formazan generated in the reaction system is proportional to the concentration of L-malic acid in the sample.

Features

• This kit has high sensitivity, a wide linear range, and requires a small sample volume. It specifically detects L-malic acid without detecting D-malic acid. With a sample volume of 20 µl, it can detect malic acid concentrations as low as 20 μM, and shows a good linear relationship within the concentration range of 20-500 μM (0.4-10 nmol). The kit provides malic acid standard solutions, allowing for the calculation of L-malic acid content in samples by setting up a standard curve.

• The detection lysis buffer provided with this kit is quite versatile. Cells or tissue samples lysed using Buffer A for Metabolic Assay included in this kit can also be used for assays with other metabolic assay kits from the same manufacturer, which also use Buffer A for Metabolic Assay. This buffer shows strong versatility and can also be used for detecting protein concentrations, performing SDS-PAGE, or Western blotting of proteins that are relatively soluble.

• The kit is flexible, fast, and widely applicable. It can be used for the detection of biological fluids such as serum, plasma, and urine from mice, rats, and humans, as well as for cell culture supernatants, tissue or cell samples, and beverages and juices. The entire process can be completed in approximately 0.5 to 1 hour. This kit is suitable for both small sample sizes and high-throughput screening in automated systems.

Storage

Store at -20ºC, valid for one year. The color development solution and substrate must be stored protected from light.

Precautions

• This kit can only detect L-malic acid or L-malic acid salts and cannot detect D-malic acid or D-malic acid salts.
• Buffer A for Metabolic Assay, malic acid detection buffer (hereafter referred to as detection buffer), color development solution, and substrate must be fully thawed and equilibrated to room temperature before use; otherwise, it may affect the results. Enzyme solutions and malic acid standard solutions should be used on ice.
• The substrate may precipitate during thawing from -20ºC. After equilibrating to room temperature, the precipitated portion will re-dissolve and will not affect the results.
• If samples such as serum are stored at 4ºC, they should not be stored for more than 2 weeks, as this may affect the accuracy of the results. Typically, serum samples should be stored at -20ºC, with -80ºC being preferable.
• To minimize errors from background caused by the diluent, the diluent for samples and standards should be selected based on the type of sample. For samples lysed with Buffer A for Metabolic Assay, use Buffer A for Metabolic Assay for dilution. For other samples such as blood, use the detection buffer for dilution.
• This product is for scientific research use by professionals only and is not for clinical diagnosis or treatment, nor for use in food or drugs, and should not be stored in ordinary residential areas.
• For your safety and health, please wear laboratory coats and disposable gloves while handling.