Lysine (LYS) Content Assay Kit (Ninhydrin, Micro Method)

Cat. No.: L1509156

GRADE & PURITY BioReagent

Descripción general

Lysine is one of the essential amino acids for the human body. It promotes development, enhances immune function, and improves the function of the central nervous system. Lysine is a basic essential amino acid. As cereal-based foods are very low in lysine content, which is easily destroyed and becomes deficient during processing, it is referred to as the first limiting amino acid.

Detection Principle:

The lysine in proteins possesses a free ε-NH₂ group, which reacts with the ninhydrin reagent to produce a blue-purple substance with a characteristic absorption peak at 570 nm. The lysine content is calculated by measuring the absorbance at 570 nm.

Detection Range: 0.125 - 8 µmol/mL

Sensitivity: 0.03125 µmol/mL

Applicable Samples: Serum; Plasma; Animal/Plant Tissues; Cells; Cell Supernatant; Bacteria

Note: It is recommended to perform a preliminary test with 2-3 samples expected to have significant differences before formal detection.

Reagents, consumables and Equipments not provided

• Microplate reader or visible spectrophotometer (capable of measuring absorbance at 570 nm)

• 96-well plate or micro glass cuvettes, adjustable pipettes and tips

• Centrifuge, water bath

• Deionized water, 95% ethanol, 60% ethanol

• Homogenizer (for tissue samples)

Procedure

1. Reagent Preparation

Reagent Name	Preparation	Notes
Extraction Buffer	Ready-to-use; equilibrate to room temperature before use.	Store at 4°C.
Assay Buffer	Ready-to-use; equilibrate to room temperature before use.	Store at 4°C.
Working Substrate	Prepare immediately before use. For 96T, add 2 mL of 95% ethanol and dissolve completely.	Unused portion can be stored at 4°C protected from light for 1 week.
Working Substrate Cofactor	Prepare immediately before use. Add all of the Assay Buffer to the Substrate Cofactor and dissolve completely. A 5-minute incubation in a 37°C water bath is recommended to facilitate dissolution.	Unused portion can be stored at 4°C for 1 week.
Working Solution	Prepare by mixing Working Substrate and Working Substrate Cofactor at a 1:1 ratio.
Standard	Prepare immediately before use. Add 1.71 mL of deionized water and dissolve completely to obtain a 40 µmol/mL standard solution.	Unused portion can be stored at 4°C protected from light for 1 week. For long-term storage, aliquot and store at -20°C protected from light, avoiding repeated freeze-thaw cycles.

Note: Chromogen and Substrate can be irritating. Personal protection is recommended during use.

2. Standard Curve Setup

Dilute the 40 µmol/mL standard with deionized water as shown in the table below to prepare standard solutions of 8, 4, 2, 1, 0.5, 0.25, and 0.125 µmol/mL.

Std. No.	Standard Volume	Deionized Water Volume (µL)	Standard Concentration (µmol/mL)
Std.1	40µL of 40 μmol/mL	160	8
Std.2	100µL of Std.1	100	4
Std.3	100µL of Std.2	100	2
Std.4	100µL of Std.3	100	1
Std.5	100µL of Std.4	100	0.5
Std.6	100µL of Std.5	100	0.25
Std.7	100µL of Std.6	100	0.125

Note: A standard curve must be prepared for each experiment. Diluted standard solutions are unstable and must be used within 4 hours.

3.Sample Preparation

Note: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for 1 month.

3.1 Animal Tissue: Weigh about 0.1g of sample, add 1 mL of Extraction Buffer, homogenize thoroughly at room temperature. Transfer to a 1.5 mL EP tube, cap tightly (to prevent moisture loss), and incubate in an 80°C water bath for 20 min for extraction. After cooling, centrifuge at 10,000 g, room temperature for 10 min. Collect the supernatant for assay.

3.2 Plant Tissue: Weigh about 0.1g of sample, add 1 mL of Extraction Buffer and crush. Sonicate at room temperature for 5 min (power 20% or 200W, pulse 3s on, 7s off, repeat 30 times). Transfer to a 1.5 mL EP tube, cap tightly, and incubate in an 80°C water bath for 20 min. After cooling, centrifuge at 10,000 g, room temperature for 10 min. Collect the supernatant.

3.3 Cells or Bacteria: Collect 5 million cells/bacteria into a centrifuge tube. Wash with cold PBS, centrifuge, discard supernatant. Add 1 mL of Extraction Buffer, sonicate as in 3.2. Transfer to EP tube, cap tightly, and incubate in 80°C water bath for 20 min. After cooling, centrifuge at 10,000 g, room temperature for 10 min. Collect the supernatant.

3.4 Cell Supernatant or Serum/Plasma: In a 1.5 mL EP tube, add 0.5 mL of sample to 0.5 mL of Extraction Buffer. Cap tightly and incubate in an 80°C water bath for 20 min. After cooling, centrifuge at 10,000 g, room temperature for 10 min. Collect the supernatant.

Note: Protein concentration determination using the Aladdin BCA Protein Assay Kit (B665595) or Ready-to-Use BCA Protein Assay Kit (R1491648) is recommended.

4. Assay Procedure

4.1 Preheat the microplate reader or spectrophotometer for at least 30 min. Set wavelength to 570 nm. Zero the spectrophotometer with deionized water.

4.2 Sample Assay (Add reagents in the following order in EP tubes):

Reagent (µL)	Blank Tube	Standard Tube	Assay Tube
Deionized Water	10	0	0
Different Conc. Std.	0	10	0
Sample	0	0	10
Working Solution	20	20	20

4.3 Mix well, cap tubes tightly (prevent moisture loss), and place in a boiling water bath for 5 min. Cool under tap water for 10 seconds. Add 170 µL of 60% ethanol, invert tubes several times. Centrifuge at 5,000 g, room temperature for 5 min. Transfer 150 µL from each tube to a 96-well plate or micro cuvette. Measure absorbance at 570 nm. Calculate ΔA_assay = A_assay - A_blank, ΔA_std = A_standard - A_blank (only 1 blank tube is needed). Measurement must be completed within 30 min after color development.

Note: Only 1-2 replicates of Blank and Standard tubes are needed. Preliminary tests with 2-3 samples of expected large differences are recommended before the formal experiment. If ΔA_assay is less than 0.002, increase sample volume appropriately. If ΔA_assay is greater than 1.8, further dilute the sample with deionized water and multiply the result by the dilution factor.

5. Calculation of Results

Note: Two formula formats are provided: a detailed derivation and a concise version. They are equivalent. The bold concise formula is recommended for final calculation.

5.1 Standard Curve Plotting: Plot standard concentration (y-axis) against ΔA_std (x-axis) to obtain the standard curve equation. Substitute ΔA_assay into the equation to calculate y (µmol/mL).

5.2 Sample Lysine Content Calculation:

(1) Calculation Based on Sample Weight

Lysine Content (μmol/g weight) = y ÷ (W ÷ V_Extraction) × n = y ÷ W × n

(2) Calculation Based on Protein Concentration

Lysine Content (μmol/mg protein) = y ÷ Cpr × n

(3) Calculation Based on Bacterial or Cell Count

Lysine Content (μmol/10⁴ cells) = y ÷ (Bacterial or Cell Count ÷ V_Extraction) × n = y ÷ 500 × n = 0.002 × y × n

(4) Calculation Based on Liquid Volume

Lysine Content (μmol/mL) = y × 2 × n

Parameter Definitions:

• W: Sample weight (g)

• V_extraction: Volume of Extraction Buffer added (1 mL)

• n: Sample dilution factor (if further diluted)

• Cpr: Protein concentration of the supernatant (mg/mL)

• 500: Total number of bacteria/cells (5 million)

• 2: Dilution factor for liquid samples, (0.5 mL + 0.5 mL) / 0.5 mL = 2

6. Result Presentation

Typical Standard Curve:

Figure 1: Standard curve for lysine content

Experimental Example: 

Figure 2: Lysine content in mouse liver and Arabidopsis thaliana determined using this kit

Precautions

This product is for research use only. Not for use in clinical diagnosis. For your safety and health, please wear a lab coat and disposable gloves during operation.

Almacenamiento y envío

Condiciones de almacenamiento de almacenamiento

Store at 2-8°C,Protected from light

Enviado en

Wet ice

Estabilidad y almacenamiento

Store at 2-8℃ long term (12 months). Store in the dark.

Contents & Storage

L1509156	Component	96T	Storage
L1509156A	Extraction Buffer	120 mL	2-8℃
L1509156B	Assay Buffer	2 mL	2-8℃
L1509156C	Substrate	1EA	2-8℃. Store in the dark.
L1509156D	Substrate Cofactor	1EA	2-8℃. Store in the dark.
L1509156E	Standard	1EA	2-8℃. Store in the dark.