Nitrate Nitrogen Content Assay Kit (Sulfanilamide, Micro Method)

Cat. No.: N1510410

GRADE & PURITY BioReagent

Descripción general

Nitrate nitrogen is the primary nitrogen source for plants. The nitrate nitrogen content within plant tissues reflects the supply of nitrate nitrogen in the soil and can serve as an indicator of soil nitrogen fertility. Determining the nitrate nitrogen content in plants is not only important for assessing plant nitrogen nutrition but also holds significant value for evaluating the quality of vegetables and processed products made from plant materials.

Detection Principle

A reducing agent converts nitrate into nitrite, which then reacts with sulfanilic acid and naphthylamine to form a rose-red azo compound. The color intensity is directly proportional to the nitrogen content within a certain range. The absorbance at 520 nm is measured using a microplate reader, and the nitrate nitrogen content of the sample is calculated based on a standard curve. This kit is primarily used to determine nitrate nitrogen content in plant tissues, serum, tissue samples, etc. It is intended for research purposes only and is not suitable for clinical diagnosis or other applications.

N1510410	Component	100T	Storage
N1510410A	Nitrate Nitrogen Standard (200 µg/mL)	1 mL	2-8℃.
N1510410B	Nitrate Nitrogen Extraction Solution	500 mL	RT.
N1510410C	Assay Buffer	500 mL	RT.
N1510410D	Sulfanilamide Mixture Powder	10 g	RT. Store in the dark.

Reagents, consumables and Equipments not provided

• Microplate reader (capable of measuring absorbance at 520 nm)
• Electronic balance, scissors, centrifuge, centrifuge tubes, test tubes, filter paper, 96-well plate
• Experimental materials: Plant tissues (e.g., soybean, corn petioles), blood, urine, etc.
• Distilled water

Procedure

1. Sample Preparation

1.1 Plant Samples

Take 0.1–0.15 g of fresh plant tissue (from normal or stressed conditions). Wash, dry, and mince into 1–2 mm pieces. Add 4 mL of Nitrate Nitrogen Extraction Solution. Vortex vigorously for 2–4 minutes. After clarification, the supernatant is the nitrate nitrogen extract. Store at 4°C for later use.

1.2 Serum and Urine Samples

Samples prepared by conventional methods can be used directly for assay with this kit. Store at 4°C for nitrate nitrogen detection.

1.3 High-Concentration Samples

If the sample contains a high concentration of nitrate nitrogen, dilute appropriately using the Nitrate Nitrogen Extraction Solution or distilled water.

2. Preparation of Nitrate Nitrogen Standard Series

Dilute the Nitrate Nitrogen Standard (200 µg/mL) with distilled water to 20 µg/mL. Then continue diluting according to the table below:

Reagent (mL)	1	2	3	4	5	6
Nitrate Nitrogen Standard (20 µg/mL)	0.05	0.1	0.2	0.3	0.4	0.5
Distilled water	0.45	0.4	0.3	0.2	0.1	0
Nitrate Nitrogen Concentration (µg/mL)	2	4	8	12	16	20

3. Loading

Set up blank tubes, standard tubes, and sample tubes according to the table below. Add solutions in the specified order, avoiding bubble formation. If the sample nitrate nitrogen concentration is too high, reduce the sample volume or dilute appropriately before assay. It is best to set up duplicate tubes for samples.

Component (mL)	Blank Tube	Standard Tube	Sample Tube
Distilled water	0.5	—	—
Nitrate Nitrogen Standard Series (Tubes 1–6)	—	0.5	—
Sample	—	—	0.5
Assay Buffer	4.5	4.5	4.5

Mix thoroughly.

Sulfanilamide Mixture Powder

0.1g

0.1g

0.1g

4. Nitrate Nitrogen Measurement

Vortex vigorously for 1 minute and let stand for 10 minutes. Filter through double-layer filter paper into a new centrifuge tube. Centrifuge at 4000 rpm for 5 minutes. Take the supernatant and centrifuge again. Transfer the supernatant to a 96-well plate. Using the blank tube as a reference to zero the instrument, measure the absorbance of the standard wells and sample wells at 520 nm using the microplate reader (recorded as A_standard and A_sample).

5. Calculation

Using the concentration (µg/mL) of the nitrate nitrogen standard series (Tubes 1–6) as the x-axis and the corresponding absorbance as the y-axis, generate a standard curve. Calculate the nitrate nitrogen content of the sample based on the absorbance of the sample well.

For plant tissue samples:

Nitrate nitrogen (µg/g) = C × V / m

• C: Nitrate nitrogen concentration of the sample obtained from the standard curve (µg/mL)

• V: Total volume of the sample extract (mL); in this method, it is 4 mL

• m: Sample weight (g)

For serum, urine, and other liquid samples:

Nitrate nitrogen (µg/mL) = C × N

• C: Nitrate nitrogen concentration of the sample obtained from the standard curve (µg/mL)

• N: Dilution factor

Precautions

1. Experimental materials should be as fresh as possible. If not assayed immediately after collection, store at 4°C.

2. The Assay Buffer is volatile; please store it sealed tightly.

3. If a microplate reader is unavailable, a standard spectrophotometer can also be used for measurement.

4. If the concentration of the sample is too high, dilute the sample with Nitrate Nitrogen Extraction Solution or distilled water and repeat the assay.

5. For your safety and health, please wear a lab coat and disposable gloves during operation.

6. After opening, use the reagent as soon as possible to avoid affecting subsequent experimental results,

Almacenamiento y envío

Condiciones de almacenamiento de almacenamiento

Store at 2-8°C,Protected from light,Room temperature

Enviado en

Wet ice

Estabilidad y almacenamiento

Each component has a shelf life of 1 year under corresponding storage conditions. N1510410A: Store at 2-8℃ long term (12 months). N1510410B, N1510410C: Store at room temperature long term (12 months). N1510410D: Store at room temperature long term (12 mon

Contents & Storage

N1510410	Component	100T	Storage
N1510410A	Nitrate Nitrogen Standard (200 µg/mL)	1 mL	2-8℃.
N1510410B	Nitrate Nitrogen Extraction Solution	500 mL	RT.
N1510410C	Assay Buffer	500 mL	RT.
N1510410D	Sulfanilamide Mixture Powder	10 g	RT. Store in the dark.