Description
Animal liver and kidneys are the main organs for amino acid metabolism. Therefore, changes in urinary amino acids best reflect the physiological state of the liver and kidneys. Additionally, amino acid levels can indicate conditions such as burns and typhoid fever. The amino acid content in plants is significant for studying nitrogen metabolism changes under different conditions and during various growth and development stages, as well as for understanding nitrogen absorption, transport, assimilation, and nutritional status in plants.Detection Principle: The α-amino group of amino acids reacts with ninhydrin hydrate to produce a blue-purple compound with a characteristic absorption peak at 570 nm. The amino acid content is calculated by measuring the absorbance at 570 nm.Detection Range: 0.625 - 40 µmol/mLSensitivity: 0.5 µmol/mLApplicable Samples: Serum (plasma), animal/plant tissues, cells, cell culture supernatants, bacteria, urineG1501758Component96TStorageG1501758AExtraction Buffer100 mL2-8℃G1501758BAssay Buffer10 mL2-8℃G1501758CSubstrate1EA2-8℃. Store in the dark.G1501758DStandard (10mg Cysteine)1EA2-8℃. Store in the dark.User-Prepared Instruments and ReagentsMicroplate reader or visible spectrophotometer (capable of measuring absorbance at 570 nm)96-well plate or micro glass cuvettes, adjustable micropipettes and tipsRefrigerated centrifuge, water bathDeionized water, EthanolHomogenizer (for tissue samples)Experimental Procedure1. Reagent PreparationReagent NameReagent PreparationNotesExtraction BufferReady-to-use; Equilibrate to room temperature before use.Store at 4°C.Assay BufferReady-to-use; Equilibrate to room temperature before use.Store at 4°C. Toxic and irritant. Perform experiments in a fume hood.SubstrateToxic and irritant. Perform experiments in a fume hood.Working SubstratePrepare before use: Dissolve in 4 mL of 95% Ethanol.Unused dissolved substrate can be stored at 4°C protected from light for one week. For long-term storage, aliquot and store at -20°C protected from light for one month. Avoid repeated freeze-thaw cycles.StandardPrepare before use: Add 2.066 mL deionized water to dissolve completely, resulting in a 40 µmol/mL stock.Unused dissolved standard can be stored at 4°C for one week. For long-term storage, aliquot and store at -20°C for one month. Avoid repeated freeze-thaw cycles.2. Standard Curve SetupDilute the 40 µmol/mL standard stock solution with deionized water to concentrations of 20, 10, 5, 2.5, 1.25, and 0.625 µmol/mL as shown in the table below.TubeStandard VolumeDeionized Water Volume (µL)Standard Concentration (µmol/mL)Std.1200µL of 40µmol/mL040Std.2100µL of Std.110020Std.3100µL of Std.210010Std.4100µL of Std.31005Std.5100µL of Std.41002.5Std.6100µL of Std.51001.25Std.7100µL of Std.61000.625Note: The standard curve must be generated with each experiment. Diluted standard solutions are unstable and must be used within 4 hours.3. Sample PreparationNote: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for up to one month.3.1 Animal TissueWeigh approximately 0.1 g of tissue. Add 1 mL of Extraction Buffer and homogenize thoroughly at room temperature. Transfer the homogenate to a 1.5 mL microcentrifuge tube. Cap tightly (to prevent moisture loss) and incubate in a boiling water bath for 15 minutes. Cool with tap water. Centrifuge at 10,000 rpm for 10 minutes at room temperature. Collect the supernatant for assay.3.2 Plant TissueWeigh approximately 0.1 g of tissue. Add 1 mL of Extraction Buffer and grind. Sonicate for 5 minutes at room temperature (power 20% or 200W, pulse 3s on, 7s off, repeat 30 times). Transfer to a 1.5 mL microcentrifuge tube. Cap tightly (to prevent moisture loss) and incubate in a boiling water bath for 15 minutes. Cool with tap water. Centrifuge at 10,000 rpm for 10 minutes at room temperature. Collect the supernatant for assay.3.3 Cells or BacteriaCollect 5 million cells or bacteria into a centrifuge tube. Wash cells with cold PBS, centrifuge, and discard the supernatant. Add 1 mL of Extraction Buffer. Sonicate for 5 minutes at room temperature (power 20% or 200W, pulse 3s on, 7s off, repeat 30 times). Transfer to a 1.5 mL microcentrifuge tube. Cap tightly (to prevent moisture loss) and incubate in a boiling water bath for 15 minutes. Cool with tap water. Centrifuge at 10,000 rpm for 10 minutes at room temperature. Collect the supernatant for assay.3.4 Serum (Plasma), Cell Culture Supernatant, Urine, and Other LiquidsPipette 0.5 mL of the liquid sample and add 0.5 mL of Extraction Buffer. Cap tightly (to prevent moisture loss) and incubate in a boiling water bath for 15 minutes. Cool with tap water. Centrifuge at 10,000 rpm for 10 minutes at room temperature. Collect the supernatant for assay.Note: If protein concentration measurement is required, use Aladdin's BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648).4. Assay Steps4.1 Preheat the microplate reader or visible spectrophotometer for at least 30 minutes. Set the wavelength to 570 nm. For spectrophotometers, zero the instrument with deionized water.4.2 Sample Measurement (Add reagents sequentially into microcentrifuge tubes as below):ReagentBlank Tube (µL)Standard Tube (µL)Test Tube (µL)Deionized Water1000Standard (various conc.)0100Sample0010Working Substrate202020Assay Buffer5050504.3 Mix well and cap the tubes tightly (to prevent moisture loss). Incubate in a boiling water bath for 5 minutes. Cool in tap water for 10 seconds. Add 120 µL of 60% ethanol to each tube and invert several times to mix. Transfer 150 µL from each tube to the corresponding wells of a 96-well plate or micro glass cuvette. Measure the absorbance at 570 nm, recorded as A blank, A standard, and A test. Calculate ΔA test = A test - A blank and ΔA standard = A standard - A blank (The blank tube only needs to be prepared once). All measurements must be completed within 30 minutes after color development. Note:It is recommended to perform a preliminary test with 2-3 samples expected to have significant differences before the formal experiment. If A <sub> test </sub> is greater than 2.0, dilute the sample further with deionized water and multiply the result by the dilution factor.Proline and hydroxyproline do not produce an absorption peak at 570 nm when reacting with ninhydrin. Therefore, the results measured at 570 nm do not include these two amino acids.5. Calculation of ResultsNote: We provide two formulas, including the derived formula and a simplified version. They are equivalent. It is recommended to use the simplified formula in bold for final calculation.5.1 Standard Curve PlottingPlot the standard concentration (y-axis) against ΔA standard (x-axis) to generate the standard curve. Substitute ΔA test into the standard curve equation to calculate y (µmol/mL).5.2 Sample Amino Acid Content Calculation(1) Based on Sample WeightAmino Acid Content (µmol/g weight) = y ÷ (W ÷ V<sub>extraction</sub>) × n = y ÷ W × n(2) Based on Protein ConcentrationAmino Acid Content (µmol/mg prot) = y ÷ Cpr × n(3) Based on Bacterial or Cell CountAmino Acid Content (µmol/10⁴ cells) = y ÷ (Count ÷ V<sub>extraction</sub>) × n = y ÷ 500 × n = 0.002 × y × n(4) Based on Liquid VolumeAmino Acid Content (µmol/mL) = y × 2 × nParameter Definitions:W: Sample weight (g)V extraction : Volume of Extraction Buffer added (1 mL)n: Sample dilution factorCpr: Protein concentration of the supernatant (mg/mL)500: Total number of bacteria or cells (5 million)2: Dilution factor for liquid samples [(0.5 mL sample + 0.5 mL Buffer) / 0.5 mL sample = 2]6. Representative ResultsTypical Standard Curve: y = 20.349x - 0.423, R² = 0.9971 Figure 1: Total Amino Acid Standard Curve Precautions1. Biochemical reagents are generally irritating, biologically toxic, etc. For your safety and health, please use appropriate biosafety precautions throughout the experiment, including wearing lab coats, masks, gloves, head covers, etc. Perform experiments in a fume hood or biosafety cabinet.2. This product is for research use only. Not for use in clinical diagnosis