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Cat. No.: ICAK-100 (for 100T)

Description

Isocitrate Assay Kit is a reagent kit based on the WST-8 colorimetric reaction, designed for the rapid and highly sensitive detection of isocitrate and its salts in biological fluids such as serum and plasma, as well as tissue, cell samples, and food products through colorimetric methods.

Isocitrate (Isocitric acid, also known as 1-Hydroxypropane-1,2,3-tricarboxylic acid) has a molecular formula of C₆H₈O₇ and a molecular weight of 192.12. It is an isomer of citric acid. Isocitrate is found in high concentrations in many fruits and vegetables and their processed products. It is commonly used as a flavoring agent or as an acidulant in beverages, and serves as a polyvalent chelating agent in both food and medical applications, as well as an intermediate in chemical processes. In industrial settings, isocitrate is used as an indicator of fruit juice quality and purity.

As a metabolite in the tricarboxylic acid cycle (TCA cycle), isocitrate is reversibly formed from citric acid by the action of aconitase. Isocitrate is oxidatively decarboxylated to α-ketoglutarate (α-KG) by isocitrate dehydrogenase (IDH) and is also split into succinate and glyoxylate by isocitrate lyase. Isocitrate plays a crucial role in various biochemical processes within the organism, including metabolism, tissue structure formation, and enzyme activity. Overall, isocitrate is essential for many biological functions within living organisms.

Components

• Buffer A for Metabolic Assay
• Isocitrate Assay Buffer
• Enzyme Solution
• Color Development Solution
• Substrate
• Isocitrate Standard Solution (100mM)

Principle

Isocitrate is oxidized to α-ketoglutarate (α-KG) by the enzyme isocitrate dehydrogenase. During this reaction, NAD+ or NADP+ is reduced to NADH or NADPH. The generated NADH or NADPH 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 produced in the reaction is directly proportional to the amount of isocitrate in the sample.

Features

• This kit has high sensitivity, a broad linear range, and requires only a small sample volume. It can detect isocitrate concentrations as low as 50 μM with a sample volume of 20 µL, and shows good linearity within the concentration range of 50 μM to 20 mM (1-400 nmol). The kit provides an isocitrate standard solution, which can be used to set up a standard curve to calculate the isocitrate content in the sample.

• This kit's lysis buffer offers a certain degree of versatility. The cell or tissue samples lysed using the BeyoLysis™ Buffer A for Metabolic Assay included in this kit can also be used for detection in other metabolic assay kits from the same manufacturer that use BeyoLysis™ Buffer A for Metabolic Assay. It is highly versatile and can also be used for protein concentration assays, SDS-PAGE, or Western blotting of easily soluble proteins.

• The kit is flexible in use, provides rapid results, and is applicable to a wide range of samples. It can be used to analyze serum, plasma, cell culture supernatant, tissue or cell samples, as well as juice and food samples from mice, rats, humans, etc. The entire process can be completed in approximately 0.5-1 hour. The kit is suitable not only for small sample analysis but also for high-throughput screening in automated systems.

Storage

Store at -20ºC. Valid for one year. Color development solution, substrate, and Isocitrate Standard Solution (100mM) must be protected from light.

Precautions

• Buffer A for Metabolic Assay, Isocitrate 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. Enzyme solution and Isocitrate Standard Solution should be kept on ice during use.
• The substrate may precipitate during thawing from -20ºC. After equilibrating to room temperature, the precipitated part will dissolve again and will not affect the test results.
• For samples such as serum stored at 4ºC, the storage time should not exceed 2 weeks, as this may affect the accuracy of the test results. Generally, serum samples should be stored at -20ºC, and -80ºC is preferable.
• To reduce background errors caused by diluent, the diluent for samples and standards should be chosen according to the type of sample. When the sample is a lysate 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 intended for scientific research use by professionals only and must not be used for clinical diagnosis or treatment, food, or drug applications, nor stored in ordinary residential settings.
• For your safety and health, please wear laboratory coats and disposable gloves while handling.