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Cat. No.: MDAAK-100 (for 100T)

Description

Malate Dehydrogenase Activity Assay Kit, Malate Dehydrogenase Assay Kit with WST-8, or MDH Assay Kit with WST-8, abbreviated as MDH Activity Detection Kit or MDH Detection Kit, is a kit based on the WST-8 colorimetric reaction. It enables rapid and highly sensitive detection of malate dehydrogenase activity in tissue or cell samples, as well as biological fluids such as serum and plasma, using a colorimetric method.

Malate dehydrogenase (MDH) is an oxidoreductase enzyme found in cells and widely distributed in animals, plants, and microorganisms. It is one of the key enzymes in biological sugar metabolism, catalyzing the interconversion between malic acid and oxaloacetic acid. MDH plays an important role in various physiological activities within cells, including mitochondrial energy metabolism, the malate-aspartate shuttle system, and the metabolism of reactive oxygen species in plants.

There are several isoenzymes of malate dehydrogenase, but in eukaryotic cells, there are two main isoforms: m-MDH, located in the mitochondria, and c-MDH, located in the cytoplasm. m-MDH primarily catalyzes the oxidation of malic acid in the mitochondria and is one of the important enzymes in the tricarboxylic acid cycle, playing a crucial role in the complete oxidation or interconversion of nutrients within the body. c-MDH, found in the cytoplasm, mainly catalyzes the reduction of oxaloacetic acid and is a key enzyme in the malate-aspartate shuttle system, which facilitates the transfer of NADH produced in glycolysis into the mitochondria for complete oxidation and energy production.

MDH plays a significant role in maintaining normal physiological functions. Clinical studies have shown that serum MDH activity significantly increases in the early stages of myocardial infarction and viral hepatitis. Additionally, elevated serum MDH activity has been observed in kidney diseases, rheumatoid and rheumatic diseases, traumatic shock, and blood system disorders. Therefore, measuring MDH activity is important for understanding related metabolic conditions and for diagnosing and preventing clinically relevant diseases.

Components

• Buffer A for Metabolic Assay
• MDH Detection Buffer
• Positive Control (100X)
• Color Development Reagent
• Substrate
• NADH

Principle

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

Features

• This kit offers high sensitivity, a broad linear range, and requires a small sample volume. It can detect malate dehydrogenase activity as low as 13 µU with a sample volume of 20 µl, and exhibits a good linear relationship within the activity range of 0.67-33.3 U/L. The kit includes a standard solution of NADH, a product of the malate dehydrogenase reaction, which can be used to set up a standard curve to calculate the malate dehydrogenase activity in the sample.

• The detection lysis buffer provided in this kit has broad applicability. Cell or tissue samples lysed using Buffer A for Metabolic Assay in this kit can also be used for detection in other metabolic assay kits produced by the same company that use Buffer A for Metabolic Assay for lysis, demonstrating strong versatility. Additionally, it can be used for protein concentration detection, SDS-PAGE, or Western blotting for some easily soluble proteins.

• This kit is flexible, with a rapid detection speed and wide applicability. It can be used to detect biological fluids such as serum and plasma from mice, rats, and humans, as well as cell culture supernatants and tissue or cell samples, with the entire process taking about 1 hour. The kit is suitable not only for small sample detection but also for high-throughput screening in automated systems.

Storage

Store at -20ºC, with a one-year shelf life. The color development reagent, substrate, and NADH must be protected from light. After preparing NADH as a solution, it should be aliquoted and stored at -80ºC.

Precautions

• The Buffer A for Metabolic Assay, MDH detection buffer (hereafter referred to as detection buffer), color development reagent, and substrate need to be completely thawed and equilibrated to room temperature before use, otherwise, it may affect the results. Other reagents should be used on ice.
• The substrate may precipitate when taken out from -20ºC during the thawing process. The precipitated part will dissolve upon reaching room temperature and will not affect the results.
• NADH is unstable; after preparing it as a solution, it should be aliquoted and stored at -80ºC, and used as soon as possible. If the standard curve is not ideal, it is likely that the standard NADH has degraded.
• For serum and other samples stored at 4ºC, the storage time should not exceed 2 weeks, as it may affect the accuracy of the results. Serum samples are typically best stored at -20ºC, with -80ºC being preferable.
• To minimize errors due to background from the diluent, the diluent for samples and standards should be selected according to the type of sample. For cell or tissue lysates prepared 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 should not be used for clinical diagnosis or treatment, nor for food or drug applications, and should not be stored in a regular residence.
• For your safety and health, please wear a lab coat and disposable gloves while handling the product.