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Cat.No: CMAK-YJL-0359 Datasheet Instruction for Use
Specification Quantities
Price $374.13–623.55
| Product Name | Total Amino Acids (T-AA) Test Kit |
| Catalog No. | CMAK-YJL-0359 |
| Description | This kit is a colorimetric assay that utilizes absorbance measurement to provide a simple, rapid, and highly sensitive method for determining the total free amino acid content in biological samples such as serum, plasma, tissues, cells, and bacteria. |
| Kit Components | Buffer A for Metabolic Assay: 15 ml T-AA Assay Buffer : 15 ml Substrate: 8 ml Cofactor: 200 µl Glycine Standard (1M): 200 µl |
| Sample Preparation | Preparation of Blood Samples: For serum samples, allow whole blood to stand at room temperature (e.g., 25°C) for 30 minutes to 2 hours. Do not shake vigorously to avoid hemolysis. Once the whole blood has naturally clotted and the serum has separated, centrifuge at 4°C at approximately 1000–2000×g for 10 minutes. Collect the yellow supernatant to obtain the serum; be careful not to aspirate the white or pale yellow precipitate. For plasma samples, anticoagulate whole blood with heparin or EDTA. Centrifuge at 4°C at approximately 1000–2000×g for 10 minutes. Collect the yellow or pale yellow supernatant to obtain plasma. Take care not to aspirate the white precipitate. Both serum and plasma must be placed on ice. If testing cannot be performed immediately, they may be aliquoted and stored short-term at –20°C or –80°C. For frozen samples, thaw them before testing and store them in an ice bath until ready for use. Ensure they are thoroughly mixed before use. Preparation of Cell or Tissue Samples: For cultured adherent cells, wash once with PBS (C0221A) and aspirate the residual liquid. For cultured suspension cells, centrifuge briefly (e.g., 100–500×g for 5 minutes) to collect the cells into a centrifuge tube, discard the supernatant, and aspirate the residual liquid. Add lysis buffer at a ratio of 100–200 μL Buffer A for Metabolic Assay per 1 million cells, pipette gently, and incubate on an ice bath for 5–10 minutes to ensure complete cell lysis. Centrifuge at 12,000×g for 3–5 minutes at 4°C, then collect the supernatant for subsequent analysis. For tissue samples, homogenize at approximately 4°C or on ice using a high-throughput tissue homogenizer (1.5/2 ml × 48) (E6618), TissueMaster™ handheld tissue homogenizer (E6600/E6607), or a glass homogenizer at a ratio of 100 μl of Buffer A for Metabolic Assay per 10 mg of tissue. Centrifuge at 4°C at approximately 12,000 × g for 3–5 minutes, and collect the supernatant for subsequent analysis. All of the above operations must be performed at 4°C or on ice. If prepared cell or tissue samples cannot be analyzed immediately, they can be stored at –20°C or –80°C. Preparation of bacterial samples: Take 1.5–4 mL of bacterial suspension with an OD₆₀₀ of 0.5–2.0, centrifuge at 5,000 × g at 4°C for 10 minutes, and discard the supernatant. Resuspend the cells in 200 μL of Buffer A for Metabolic Assay; briefly vortex to ensure complete resuspension. Perform homogenization at low temperatures (e.g., approximately 4°C or in an ice bath) using the TissueMaster™ High-Throughput Tissue Homogenizer (1.5/2 ml × 48) (E6618), the TissueMaster™ Handheld Tissue Homogenizer (E6600/E6607), or a glass homogenizer. Centrifuge at 12,000–16,000 × g (16,000 × g is preferred) at 4°C for 5 minutes. Carefully aspirate the supernatant for subsequent assays. If prepared bacterial samples cannot be analyzed immediately, they may be stored at –20°C or –80°C. |
| Sample Analysis | Preparation of the glycine standard curve. Take 5 μl of Glycine Standard (1 M), add 95 μl of Buffer A for Metabolic Assay or T-AA Assay Buffer, mix well, and prepare a 50 mM glycine standard solution. Dispense 0, 0.2, 0.4, 0.8, 2, 4, 8, and 20 μL of the 50 mM glycine standard solution into the standard wells of a 96-well plate, and make up to 20 μL with Buffer A for Metabolic Assay or T-AA Assay Buffer. The resulting standard curve concentrations are 0, 0.5, 1, 2, 5, 10, 20, and 50 mM. Dispense 1–20 μL of sample or diluted sample into the sample wells of the 96-well plate, and add Buffer A for Metabolic Assay or T-AA Assay Buffer to the sample wells as needed to bring the volume to 20 μL. At the same time, set up wells containing only Buffer A for Metabolic Assay or T-AA Assay Buffer as blank controls. To ensure sample values fall within the standard curve range, it is recommended to conduct a preliminary experiment by preparing samples at multiple dilution factors to determine the approximate amino acid concentration. If the values fall outside the standard curve range, adjust the dilution factor or the sample volume. If testing cell or tissue lysates prepared with Buffer A for Metabolic Assay, use Buffer A for Metabolic Assay for dilution; if testing samples such as blood or supernatants that do not require lysis, use T-AA Assay Buffer for dilution. Let the total dilution factor of the sample be denoted as n (for example, in this step, the sample was diluted 5-fold; if 2 µL of the “diluted sample” was added, then n = 5 × 20 / 2 = 50). For complex samples such as serum or tissue lysate supernatants, it is recommended to perform preliminary experiments with multiple dilution factors. At an appropriate dilution factor, the sample signal value and the protein content in the sample typically exhibit a good linear relationship. Based on the results of the preliminary experiments, select an appropriate dilution factor for subsequent measurements; otherwise, even if the signal values fall within the linear range of the standard curve, ideal detection results may not be obtained. Add 80 µL of T-AA reaction buffer to each well, mix well, and incubate at 25°C in the dark for 20 minutes. If the absorbance is too low, the reaction time may be extended appropriately, for example, to 30 or 45 minutes. As the reaction time increases, the signal values in the high-concentration standard wells may reach a plateau, causing them to fall outside the linear range of the standard curve. In this case, the outlier data points can be discarded, and only the data within the linear range should be used to fit the standard curve. Measure the absorbance at 570 nm. Plot a standard curve and calculate the total amino acid concentration A (mM) in the sample. If the background signal of the sample is relatively high, subtract the background signal from the sample’s signal value. The formula for calculating the total amino acid concentration is as follows: C (mM) = A × n, where A is the total amino acid concentration (mM) determined from the standard curve; n is the total dilution factor of the sample. |
| Storage and Transportation | Store at -20°C. Valid for one year. The Substrate and Cofactor must be stored away from light. |
| Precautions | This kit is commonly used to determine the total free amino acid content in samples. If you need to detect all amino acids in proteins or peptides, you must first hydrolyze the proteins or peptides into free amino acids before performing the assay. T-AA Assay Buffer and Substrate must be equilibrated to room temperature before use; otherwise, test results may be affected. All other solutions should be handled on ice. To minimize errors caused by background from diluents, the diluents for samples and standards should be selected based on the type of sample. When the sample is a bacterial, cellular, or tissue lysate prepared using Buffer A for Metabolic Assay, it should be diluted with Buffer A for Metabolic Assay; when the sample is prepared using other lysis buffers, it should be diluted with the corresponding lysis buffer; when the sample is blood or other types of samples, it is recommended to use T-AA Assay Buffer for dilution. If samples such as serum or plasma are stored at 4°C, the storage period must not exceed 2 weeks; otherwise, the accuracy of the test results may be compromised. Typically, serum samples should be stored at -20°C, and storage at -80°C is preferable. T-AA Assay Buffer and Cofactor are harmful or irritating to humans and may exhibit specific organ toxicity (e.g., respiratory or reproductive) or teratogenic and carcinogenic effects. Handle with care and ensure effective protective measures to avoid direct contact with the body or inhalation. Cofactor is corrosive. Handle with care and ensure effective protection to avoid direct contact with the body. Take care to prevent corrosion of other materials. This product is intended solely for scientific research by qualified professionals. It must not be used for clinical diagnosis or treatment, nor in food or pharmaceuticals, and must not be stored in residential homes. For your safety and health, please wear a lab coat and disposable gloves when handling this product. |
For research use only, not for clinical use.
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