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Cat. No.: BCAAAK-100 (for 100T)

Description

Branched Chain Amino Acid Assay Kit, or BCAA Assay Kit with WST-8, is a reagent kit based on the WST-8 colorimetric reaction. It allows for rapid and highly sensitive detection of branched-chain amino acids in biological fluids such as serum, plasma, and urine, as well as in tissue and cell lysates, and cell culture supernatants, using a colorimetric method.

Branched-chain amino acids (BCAA) refer to the three amino acids with branched side chains: leucine, valine, and isoleucine. These three amino acids are essential amino acids for the human body. Leucine is the most abundant amino acid in proteins and is a major product of protein metabolism in the diet or protein breakdown in tissues. Branched-chain amino acids account for 35% of the essential amino acids in muscle proteins and 40% of essential amino acids in mammals. They are not only raw materials for protein synthesis but also have specific biological functions and can serve as an energy source for the body. They are widely used in medicine, food, and feed industries.

Branched-chain amino acids have various metabolic and physiological functions. Metabolically, BCAAs are involved in protein synthesis, signal transduction, and glucose metabolism. Physiologically, they play important roles in the immune and nervous systems. BCAAs are effectively broken down by dehydrogenases and decarboxylases expressed by immune cells and are essential for lymphocyte growth, proliferation, and cytotoxic T lymphocyte activity. BCAAs share the same transport proteins in the brain with aromatic amino acids, playing roles in protein synthesis, neurotransmitter synthesis, and energy metabolism. As essential amino acids, BCAAs cannot be synthesized in the human body but can be catabolized by certain enzymes. Many enzymes acting on these BCAAs can use these three amino acids as substrates, thereby regulating their levels. A change in the level of one branched-chain amino acid usually leads to similar directional and quantitative changes in the other two amino acids.

Components

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

Principle

Branched-chain amino acids are oxidized to α-ketoisocaproate (KIC) by branched-chain amino acid dehydrogenases (Leucine Dehydrogenase, Isoleucine Dehydrogenase, and Valine Dehydrogenase, Leu/Ile/Val Dehydrogenase). During this reaction, NAD+ is reduced to NADH. The resulting NADH 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 peak absorbance around 450 nm. The amount of Formazan generated in the reaction system is directly proportional to the content of branched-chain amino acids in the sample.

Features

• This kit offers high sensitivity, a wide linear range, and requires minimal sample volume. It can detect branched-chain amino acids at concentrations as low as 20 μM with a sample volume of 20 μl. The kit demonstrates a good linear relationship within a concentration range of 20 μM to 2000 μM (0.4-40 nmol). A standard leucine solution is provided to facilitate the creation of a standard curve, which allows for the calculation of branched-chain amino acid content in the samples.

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

• This kit is flexible in use, fast in detection, and has a wide range of applications. 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 supernatant, tissue or cell samples. The entire process can be completed in about 0.5 to 1 hour. The kit is suitable not only for the detection of small sample sizes but also for high-throughput screening in automated systems.

Storage

Store at -20ºC. Valid for one year. Color development solution and substrate must be protected from light.

Precautions

• Buffer A for Metabolic Assay, the BCAA detection buffer (hereinafter referred to as detection buffer), color development solution, and substrate need to be completely thawed and equilibrated to room temperature before use, as otherwise, it may affect the detection results. Enzyme solution and leucine standard solution should be kept on ice during use.
• The substrate may precipitate during thawing from -20ºC. After equilibrating to room temperature, any precipitated parts will redissolve and will not affect the detection results.
• If serum or other samples are stored at 4ºC, the storage time should not exceed 2 weeks, as it may affect the accuracy of the results. Typically, serum samples should be stored at -20ºC, with -80ºC being preferable.
• To reduce errors caused by background from diluents, the diluent for samples and standards should be chosen based on the type of sample. When the sample is a cell or tissue lysate prepared using Buffer A for Metabolic Assay, it should be diluted with Buffer A for Metabolic Assay. For other samples, such as blood, the detection buffer should be used for dilution.
• This product is for scientific research use only by professionals and must not be used for clinical diagnosis or treatment, for food or pharmaceuticals, or stored in ordinary residential settings.
• For your safety and health, please wear laboratory clothing and disposable gloves while handling the products.