Soil Nitrate Reductase (S-NR) Activity Assay Kit - Microplate Method

Product Information

Product code: 67112

Soil nitrate reductase (S-NR) catalyzes the reduction of nitrate to nitrite in soil and is a key enzyme in soil nitrate nitrogen reduction. Measuring S-NR activity is useful for studies related to rational fertilization and reducing nitrogen loss.

S-NR catalyzes the following reaction:

NO₃^-+ NADH + H⁺→ NO₂^-+ NAD⁺+ H₂O

Under acidic conditions, the generated nitrite reacts with p-aminobenzenesulfonic acid and α-naphthylamine to quantitatively form a red azo compound. This compound has a maximum absorbance peak at 540 nm.

Actual readings may vary depending on the testing instrument and conditions.

Package Contents

Quality and Safety Information

Shipping and Storage

Instructions for Use

1. Sample Processing

Naturally air-dry fresh soil samples or dry them in a 37°C oven. Pass the dried samples through a 30-50 mesh sieve.

2. Reagent Preparation

• Reagent III: If crystals precipitate, dissolve before use in a 60°C-90°C water bath.
• Reagent V: Standard stock solution, 1 mL. Store at -20°C.
• 0.1 μmol/mL standard solution: Dilute Reagent V 100 times before use. Add 0.1 mL of Reagent V and dilute with distilled water to a final volume of 10 mL.

3. Assay Procedure

Add the following reagents to capped centrifuge tubes:

1. Mix well, cover the tubes, and incubate in a 37°C water bath for 24 h.
2. Centrifuge at 8000 × g, 25°C for 10 min, and collect the supernatant.
3. Add the following reagents to new tubes:

4. Mix well and allow color development at 25°C for 20 min.
5. Centrifuge at 4000 × g, 25°C for 10 min.
6. Transfer 200 μL of supernatant to a 96-well plate.
7. Zero the instrument with distilled water and read the absorbance of each tube at 540 nm.

The standard tube and blank tube only need to be measured once. Prepare one control tube for each assay tube.

Before formal measurement, perform a preliminary test using 2 samples.

4. Activity Calculation

Unit definition: The amount of enzyme that produces 1 μmol NO₂^-per gram of soil sample per day is defined as one S-NR activity unit.

S-NR (μmol/d/g soil sample) = C_standard× (A_{assay tube}- A_{control tube}) ÷ (A_{standard tube}- A_{blank tube}) × V_{total reaction}÷ W ÷ T

S-NR (μmol/d/g soil sample) = 0.5 × (A_{assay tube}- A_{control tube}) ÷ (A_{standard tube}- A_{blank tube})

Precautions

1. Before formal measurement, select 2-3 samples with large expected differences for a preliminary test.
2. Instruments and supplies required but not provided: microplate reader, water bath, benchtop centrifuge, adjustable pipettes, 96-well plate, and distilled water.
3. A 100T kit can test 48 samples.

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