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Cat. No.: FSAK-100 (for 100T)

Description

Fumarase Activity Assay Kit, Fumarase Assay Kit with WST-8, Fumarate Hydratase Assay Kit with WST-8, or FH Assay Kit with WST-8 is a kit based on the WST-8 colorimetric reaction, which allows for the rapid and highly sensitive detection of fumarase activity in cell, tissue, or blood samples using colorimetric methods.

Fumarase, also known as fumarate hydratase (FH), is an enzyme that catalyzes the reversible hydration reaction between fumarate and L-malate. It is widely present in animals, plants, and microorganisms. Fumarase exists in two forms: mitochondrial and cytosolic. The mitochondrial form is a key enzyme in the tricarboxylic acid cycle (TCA cycle), where it facilitates a transition step that produces energy in the form of NADH. The cytosolic form plays an important role in amino acid and fumarate metabolism. Fumarase is widely present in fetal and adult tissues, with higher expression levels in skin, parathyroid glands, lymph nodes, and colon. Mutations affecting fumarase production and development have led to several fumarase-related diseases in humans, including benign mesenchymal tumors of the uterus, leiomyomatosis, renal cell carcinoma, and fumarase deficiency. Human fumarase deficiency can cause severe health issues, such as fetal brain abnormalities, hypotonia, and renal cell carcinoma. Therefore, accurate measurement of fumarase activity is crucial for understanding related metabolic conditions and for the prevention, diagnosis, and mechanistic research of fumarase deficiency and related diseases.

Components

• Buffer A for Metabolic Assay
• Fumarase Detection Buffer
• Enzyme Solution
• Positive Control (100X)
• Color Development Solution
• Substrate
• NADH

Principle

Fumarase converts fumarate into L-malate, which is then oxidized to oxaloacetic acid (OAA) by malate dehydrogenase (MDH). During this reaction, NAD+ is reduced to NADH. The resulting NADH then reduces WST-8 to an orange-yellow formazan in the presence of the electron coupling reagent 1-mPMS (1-Methoxy-5-methylphenazinium methyl sulfate), with a maximum absorption peak around 450 nm. The amount of formazan generated in the reaction is directly proportional to the fumarase activity in the sample.

Features

• This kit offers high sensitivity, a wide linear range, and requires a small sample volume. It can detect fumarase activity as low as 13 µU with a sample volume of 20 µl, and shows good linearity within the activity range of 0.67-33.3 U/L. The kit includes a standard solution of NADH, a product of fumarase activity, which can be used to create a standard curve for calculating fumarase activity in the samples.

• The detection lysis solution provided in this kit has a certain level of versatility. Cell or tissue samples lysed with the lysis solution included in this kit can also be used for testing with other metabolic assay kits produced by the same manufacturer, which use the same lysis solution, demonstrating strong versatility. Additionally, it can be used for protein concentration assays, SDS-PAGE, or Western blotting for proteins that are easily soluble.

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

Storage

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

Precautions

• Buffer A for Metabolic Assay, Fumarase Detection Buffer (hereafter referred to as detection buffer), color development solution, and substrate need to be fully thawed and equilibrated to room temperature before use, or it may affect the test results. Other reagents should be kept on ice during use.
• The substrate may precipitate upon thawing from -20ºC, but the precipitate will dissolve upon equilibration to room temperature and will not affect the test results.
• Serum and other samples should not be stored at 4ºC for more than 2 weeks, as this may affect the accuracy of the results. Serum samples are generally recommended to be stored at -20ºC, with -80ºC being preferable.
• This product is intended for scientific research by professionals only and should not be used for clinical diagnosis or treatment, in food or pharmaceuticals, or stored in ordinary residential areas.
• For your safety and health, please wear lab coats and disposable gloves during operation.