As the fundamental building blocks of proteins, amino acids play pivotal roles in biological metabolism. The liver and kidneys serve as the primary organs for amino acid metabolism in animals. Variations in urinary amino acid levels reflect the body’s overall amino acid metabolic profile, as well as the physiological status of the liver and kidneys. In plants, measurements of endogenous amino acid concentrations are of great significance for investigating nitrogen metabolic shifts across developmental stages, root physiology, and the uptake, translocation, assimilation of nitrogen nutrients, alongside overall plant nutritional status.
Under heating and weakly acidic conditions, the α-amino group of amino acids reacts with ninhydrin hydrate to form a blue-violet chromogenic complex, which exhibits a characteristic absorption peak at 570 nm. Amino acid concentrations can therefore be quantified by measuring changes in absorbance.
Components
A1515867 | Components | Appearance | 100T | Storage | Quantity Per Test |
A1515867A | Extraction Buffer | Liquid | 120 mL | 2-8℃ | 300 μL |
A1515867B | Assay Buffer | Liquid | 60 mL | 2-8℃. | 500 μL |
A1515867C | Ninhydrin | Solid | 2 EA | 2-8℃. Store in the dark. | 200 μL |
A1515867D | Ascorbic acid | Solid | 2 EA | 2-8℃. Store in the dark. | 500 μL |
A1515867E | Standard | Solid | 1 EA | 2-8℃. Store in the dark. | 250 μL |
Note:
Components A and B are corrosive and volatile. Handle with caution and store in tightly sealed containers to prevent volatilization.
Components C and D are toxic and irritant. Appropriate personal protective equipment shall be worn during handling.
Reagents, consumables and Equipments not provided
Spectrophotometer (able to measure absorbance at 570 nm)
Centrifuge tubes or test tubes, cuvettes
Centrifuge, water bath, mortar or homogenizer, magnetic stirrer or ultrasonic device
Ammonia-free distilled water or deionized water, 95% ethanol, 60% ethanol
Instructions for Use
1. Extraction of Amino Acids
Note: Fresh samples are recommended. Samples can be stored at -80 °C for up to 1 month if the assay cannot be performed immediately.
① Animal tissue
Weigh approximately 0.1 g animal tissue sample and add 1 mL extraction solution. Homogenize thoroughly at room temperature, then transfer the mixture into a 1.5 mL EP tube (screw-cap EP tubes are recommended). Tighten the cap to avoid water loss, and incubate the tube in a boiling water bath for 15 min. Cool the tube with tap water, centrifuge at 10,000 g for 10 min at room temperature, and collect the supernatant for subsequent measurement.
② Plant tissue
Weigh approximately 0.1 g plant tissue sample, add 1 mL extraction solution and grind the tissue. Perform ultrasonic lysis at room temperature for 5 min (power: 20% or 200 W; pulse setting: 3 s sonication, 7 s interval, 30 cycles total). Transfer the homogenate into a 1.5 mL screw-cap EP tube. Seal the tube tightly to prevent water evaporation, then heat in a boiling water bath for 15 min. Cool with tap water, centrifuge at 10,000 g for 10 min at room temperature, and retain the supernatant for testing.
③ Bacteria or cultured cells
Collect 5×10⁶ bacterial or mammalian cells. Wash the pellet with pre-cooled PBS, centrifuge at 800 g for 2 min, and discard the supernatant. Add 1 mL extraction solution and lyse the pellet by sonication at room temperature for 5 min (power: 20% or 200 W; pulse setting: 3 s sonication, 7 s interval, 30 cycles total). Transfer the lysate to a 1.5 mL screw-cap EP tube, seal tightly to avoid water loss, and heat in boiling water for 15 min. After cooling under tap water, centrifuge at 10,000 g for 10 min at room temperature, and collect the supernatant to be assayed.
④ Liquid samples: serum (plasma), cell culture supernatant, urine, etc.
Pipette 0.5 mL liquid sample and mix with 0.5 mL extraction solution in a 1.5 mL screw-cap EP tube. Tighten the cap to prevent water evaporation, then incubate in a boiling water bath for 15 min. Cool the tube with tap water, perform centrifugation at 10,000 g for 10 min at room temperature, and collect the supernatant for measurement.
2. Reagent Preparation
2.1 Preparation of Ninhydrin Chromogenic Solution
Dissolve one vial of ninhydrin hydrate completely in 10 mL of 95% ethanol to prepare the ninhydrin chromogenic solution. Unused reagent can be stored at 4℃ away from light for up to one week.
Note: Ninhydrin hydrate is toxic and irritating. Appropriate personal protective equipment must be worn during handling.
2.2 Preparation of Vitamin C Working Solution
Accurately dissolve one vial of vitamin C in 30 mL assay buffer and mix thoroughly. If the solution cannot be used up in one experiment, aliquot it into small tubes and store at -20℃; it remains valid for one week after preparation. The kit contains excess vitamin C powder and corresponding working solution volume.
2.3 Preparation of Amino Acid Standard Stock Solution
Weigh out 10 mg amino acid standard powder and dissolve fully in 1.332 mL deionized water to obtain a 7.5 mg/mL amino acid standard stock solution. Unused stock solution can be stored at 4℃ protected from light for up to one month.
Dilute the 7.5 mg/mL standard stock solution stepwise according to the table below. A standard curve shall be generated with serial diluted standards in every single assay. The diluted standard solutions are unstable and must be used within 4 hours after dilution.
No. | Standard (μL) | Extraction Buffer（μL） | Concentration (μg/mL) |
1 | 25 µL of 7.5 mg/mL | 975 | 187.5 |
2 | 200 µL of 187.5 μg/mL | 200 | 93.75 |
3 | 200 µL of 93.75 μg/mL | 200 | 46.875 |
4 | 200 µL of 46.875 μg/mL | 200 | 23.4375 |
5 | 200 µL of 23.4375 μg/mL | 200 | 11.7188 |
6 | 200 µL of 11.71875 μg/mL | 200 | 5.8594 |
Blank | 0 | 200 | 0 |
3. Sample Assay
3.1 Preheat the spectrophotometer for more than 30 min and set the wavelength to 570nm.
3.2 Carry out the operations in 2 mL EP tubes (screw-cap EP tubes are recommended) according to the table below:
Reagent | Blank Tube (μL) | Standard Tube (μL) | Test Tube (μL) |
Extraction Buffer | 100 | 0 | 0 |
Standard | 0 | 100 | 0 |
Sample | 0 | 0 | 100 |
Ninhydrin Chromogenic Solution | 200 | 200 | 200 |
Vitamin C Working Solution | 500 | 500 | 500 |
3.3 Mix thoroughly and tighten the tube cap to prevent water loss. Incubate in a boiling water bath for 5 min, cool in an ice bath for 30 s, add 1.2 mL of 60% ethanol, and invert the EP tube repeatedly several times.
3.4 Zero the instrument with the blank tube, then measure the absorbance at 570 nm, recorded as Ablank,Astandard and Asample respectively. The absorbance must be measured within 30 min after color development.
4.1 Data Processing
Calculate ΔAsample = Asample − Ablank
ΔAstandard = Astandard − Ablank
4.2 Standard Curve Plotting
Plot the standard curve with standard concentration as the X-axis and ΔAstandard as the Y-axis.
4.3 Calculation of Amino Acid Content in Samples
(1) Calculation based on sample weight
Amino acid content (μg/g) = Y ÷ (W ÷ V₍extraction₎) × n = Y ÷ W × n
(2) Calculation based on bacterial or cell count
Amino acid content (μg/10⁴ cells) = Y ÷ (N ÷ V₍extraction₎) × n = Y ÷ N × n
(3) Calculation based on liquid volume
Amino acid content (μg/mL) = Y × 2 × n
(4) Calculation based on protein concentration
Amino acid content (μg/mg prot) = Y ÷ C₍prot₎ × n
Parameter Definition
W: Sample weight, g
V₍extraction₎: Volume of extraction solution added, fixed at 1 mL
n: Dilution factor of the sample
C₍prot₎: Protein concentration of supernatant, mg/mL
N: Number of bacteria or cells, in units of 10⁴ (e.g., if the cell count is 5×10⁶, N = 500)
2: Dilution factor for liquid samples: (0.5 mL sample + 0.5 mL extraction solution) ÷ 0.5 mL sample = 2
Precautions
1. Before formal testing, it is recommended to conduct a pilot test with 2–3 samples with expected large differences in amino acid levels.
2. For tissue and cell samples, protein concentration measurement can be used to normalize results across samples. Recommended kits: Aladdin BCA Protein Quantitation Kit (Cat. No. B665595) or Ready-to-Use BCA Protein Quantitation Kit (Cat. No. R1491648).
3. Proline and hydroxyproline produce no characteristic absorption peak at 570 nm after reaction with ninhydrin, so the measurement at 570 nm does not include these two amino acids. Specialized kits are recommended for their separate detection:
Cat. No. P1515888: Proline (PRO) Content Assay Kit (Ninhydrin Colorimetric Method)
Cat. No. P1515887: Proline (PRO) Content Assay Kit (Ninhydrin Microplate Method)
Cat. No. H1515817: Hydroxyproline (HYP) Content Assay Kit (Ehrlich Microplate Method)
4. Components A and B are corrosive and volatile. Handle with care and store hermetically sealed to avoid volatilization. Components C and D are toxic and irritant; adequate personal protective equipment shall be worn during operation.
5. Operators are advised to generate their own standard curve for higher test accuracy. If no self-generated curve is available, calculate results using the typical standard curve equation provided in the Result Demonstration section.
6. Biochemical assay reagents are generally irritant and biologically toxic. For your safety and health, full biosafety protection is required throughout the experiment, including lab coat, respirator mask, gloves and hair cap. All operations shall be performed inside a fume hood or biosafety cabinet.
7. This kit is intended for research use only and shall not be applied for clinical diagnosis or other medical purposes.
