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Cat. No.: BCAAAK-100 (for 100T)

Description

Isocitrate Dehydrogenase Assay Kit, also known as the Isocitrate Dehydrogenase Assay Kit with WST-8, IDH Activity Assay Kit with WST-8, or IDH Assay Kit with WST-8, is a kit used for the rapid and highly sensitive detection of isocitrate dehydrogenase activity in tissue or cell samples, biological fluids such as serum and plasma, and cell culture supernatants through a colorimetric assay based on the WST-8 chromogenic reaction. This kit can be used to detect NAD+-dependent IDH, NADP+-dependent IDH, or the total enzyme activity of both.

Isocitrate dehydrogenase (IDH or ICDH) is an enzyme widely present in animals, plants, microorganisms, and cultured cells. It catalyzes the oxidative decarboxylation of isocitrate to produce α-ketoglutarate (α-KG) and CO2 and reduces NAD+ or NADP+ to NADH or NADPH. In living organisms, there are two types of isocitrate dehydrogenase: NAD+-dependent isocitrate dehydrogenase (NAD+-IDH), which uses NAD+ as a coenzyme, and NADP+-dependent isocitrate dehydrogenase (NADP+-IDH), which uses NADP+ as a coenzyme. In the human body, there are three types of isocitrate dehydrogenase: IDH1, IDH2, and IDH3. IDH1 is found in the cytoplasm, while IDH2 and IDH3 are found in mitochondria. IDH1 and IDH2 are both NADP+-dependent, while IDH3 is NAD+-dependent. Therefore, it is not reasonable to use the detection of NAD+-dependent IDH and NADP+-dependent IDH to distinguish mitochondrial and cytoplasmic IDH activity.

By pre-separating mitochondria and cytoplasm, this kit can be used to detect the enzyme activity levels of human IDH1, IDH2, and IDH3.

Isocitrate dehydrogenase is an important oxidoreductase with decarboxylation activity in the body, primarily distributed in tissues and organs such as the liver, skeletal muscle, and kidneys. Clinically, isocitrate dehydrogenase is often used in the diagnosis of various diseases, such as acute and chronic hepatitis, liver cirrhosis, liver cancer, cholecystitis, bile duct obstruction, pancreatitis, and more. In particular, elevated serum isocitrate dehydrogenase is often a signal of liver metastasis in malignant tumors. In cases of extensive hepatocyte necrosis, serum isocitrate dehydrogenase levels may decrease, indicating a poor prognosis. Therefore, detecting isocitrate dehydrogenase activity is important for understanding related metabolic conditions and the diagnosis, prevention, and treatment of clinical diseases.

As a rate-limiting enzyme in the tricarboxylic acid cycle (TCA cycle), isocitrate dehydrogenase plays a crucial role not only in energy metabolism and amino acid and vitamin synthesis but also in regulating carbon flux distribution between the TCA cycle and the glyoxylate shunt. Consequently, isocitrate dehydrogenase is considered one of the best models for studying the relationship between protein structure and function, enzyme catalytic and regulatory mechanisms, and the adaptive relationship between protein functional evolution and metabolic pathways.

Components

• Buffer A for Metabolic Assay
• IDH Detection Buffer
• Positive Control (100X)
• Color Development Solution
• Substrate
• NAD+
• NADP+
• NADPH

Principle

Under the action of isocitrate dehydrogenase, isocitrate is oxidatively decarboxylated to produce α-ketoglutarate (α-KG) and CO2, during which NAD+ or NADP+ is reduced to NADH or NADPH. The generated NADH or NADPH reduces WST-8 to produce an orange-yellow formazan under the action 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 system is directly proportional to the activity of isocitrate dehydrogenase in the sample.

Features

• This kit offers high sensitivity, a wide linear range, and requires a small sample volume. The kit can detect isocitrate dehydrogenase activity as low as 13 µU when using a sample volume of 20 µl and shows a good linear relationship within the activity range of 0.67-33.3 U/L (0.013-0.666 mU). The kit provides a standard solution of NADPH, the product of isocitrate dehydrogenase activity, which can be used to set up a standard curve to calculate the enzyme activity in the sample.

• The detection lysis buffer provided in this kit has a certain degree of versatility. Cell or tissue samples lysed using the Buffer A for Metabolic Assay included in this kit can also be used for detection with other metabolic assay kits from the same manufacturer that use Buffer A for Metabolic Assay for lysis, demonstrating strong compatibility. Additionally, it can be used to measure protein concentration, perform SDS-PAGE, or conduct Western blotting for proteins that are easily soluble.

• The kit offers rapid detection and broad applicability. It can be used to test biological fluids such as serum, plasma, and urine from mice, rats, humans, as well as cell culture supernatants and tissue or cell samples. The entire process can be completed in about 0.5-1 hour. This kit is suitable not only for small sample detections but also for high-throughput screening automated systems.

• This kit offers flexible usage. It provides both coenzymes NAD+ and NADP+, allowing it to be used for detecting NAD+-IDH, NADP+-IDH, or total IDH. If you need to measure NAD+-IDH activity, only NAD+ should be added to the color development working solution. For NADP+-IDH activity, only NADP+ should be added. To measure the activity of both types of IDH, both NAD+ and NADP+ need to be added. It is important to note that NAD+, NADP+, NADH, and NADPH in the samples may cause interference, so proper sample controls should be set.

Storage

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

Precautions

• Buffer A for Metabolic Assay, IDH Detection Buffer (hereafter referred to as Detection Buffer), color development solution, and substrate need to be completely thawed and equilibrated to room temperature before use, otherwise, it may affect the detection results. Other reagents should be handled on ice during use.
• Samples such as serum should not be stored at 4ºC 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.
• This product is for scientific research use only by professionals and should not be used for clinical diagnosis or treatment, nor for food or drug applications. It should not be stored in a regular residential setting.
• For your safety and health, please wear laboratory coats and disposable gloves while handling the product.