Soil Nitrite Reductase(S-NiR) Activity Assay Kit
Spectrophotometry
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The Soil Nitrite Reductase (S-NiR) Activity Assay Kit is primarily used to measure the activity of nitrite reductase in soil samples. This enzyme plays a critical role in the nitrogen cycle, specifically in the reduction of nitrite to nitric oxide, which is a key step in denitrification processes. By assessing S-NiR activity, researchers can evaluate the efficiency of nitrogen transformation in soils, which is essential for understanding soil fertility and nutrient management.
This kit is particularly valuable in agricultural research, where optimizing nitrogen use efficiency is crucial for crop productivity and minimizing environmental impacts such as nitrate leaching and greenhouse gas emissions. It is also employed in environmental studies to monitor soil health and the impact of agricultural practices on microbial activity. Additionally, the assay helps in assessing the effects of fertilizers, pesticides, and other soil amendments on microbial enzyme functions, aiding in the development of sustainable farming practices.
Overall, the Soil Nitrite Reductase Activity Assay Kit serves as a vital tool for advancing soil science, agriculture, and environmental conservation efforts.
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Soil Nitrite Reductase Activity Assay Kit - Spectrophotometric Method
Product Information
Product code: 67051
Soil nitrite reductase is a key enzyme in denitrification. It participates in the reduction of nitrite to NO, and its activity reflects nitrogen transformation efficiency during biodegradation. This assay provides a basis for studying nitrogen transformation patterns.
Nitrite reductase reduces NO2-to NO, decreasing the amount of NO2-in the sample that participates in the diazotization reaction to form a purplish-red compound. Therefore, the change in absorbance at 540 nm reflects soil nitrite reductase activity.
Reference example: Sample: forest soil. OD540 nm: soil-free tube 1.508; blank tube 1: 0.005; control tube: 0.043; measurement tube: 1.143/1.159.
Actual readings may vary depending on the testing instrument and test conditions. These data are for reference only.
Package Contents and Storage
| Code | Component | Quantity | Storage Conditions |
|---|---|---|---|
| 67051.1 | Reagent 1 | 6 mL | 2-8°C, protect from light |
| 67051.2 | Reagent 2 | 0.12 g | 2-8°C |
| 67051.3 | Reagent 3 | 12 mL | 2-8°C |
| 67051.4 | Reagent 4 | 25 mL | 2-8°C, protect from light |
| 67051.5 | Reagent 5 | 25 mL | 2-8°C, protect from light |
| 67051.6 | Standard | 1 mL | 2-8°C, protect from light |
| 67051.m | Instructions | 1 copy | - |
Quality and Safety Information
| Raw Material or Packaging Name | Quality Standard | Main Toxicity |
|---|---|---|
| Reagent 1 | -- | -- |
| Reagent 2 | -- | -- |
| Reagent 3 | -- | -- |
| Reagent 4 | -- | -- |
| Reagent 5 | -- | -- |
| Standard | -- | -- |
Transportation and Storage
Transportation: This product is transported with ice packs.
Storage: Store according to the instructions in this manual. Shelf life: 180 days.
Instructions for Use
1. Sample Processing
Air-dry fresh soil samples naturally or in a 37°C oven, then pass through a 30-50 mesh sieve.
2. Reagent Preparation
- Reagent 2: Before use, add 12 mL distilled water to dissolve. Store at 4°C for up to 2 weeks or at -20°C for up to one year. After use, promptly store at 4°C or -20°C.
- Reagent 3: If precipitation occurs, heat at 60-80°C to dissolve.
- Reagent 4: If precipitation occurs, heat at 60-80°C to dissolve.
- Standard: 10 μmol/mL sodium nitrite.
- Working solution: Before use, mix Reagent 4 and Reagent 5 at a 1:1 ratio according to the required amount. Prepare fresh before use. After preparation, the working solution is valid for 48 h at 4°C.
3. Operating Procedure
- Preheat the spectrophotometer for 30 min or longer. Set the wavelength to 540 nm and zero with distilled water.
- Dilute the 10 μmol/mL standard solution with distilled water to prepare 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.0078125, and 0.001953125 μmol/mL standard solutions for testing.
Standard Solution Dilution
| Serial No. | Concentration Before Dilution (μmol/mL) | Standard Solution Volume (μL) | Distilled Water Volume (μL) | Concentration After Dilution (μmol/mL) |
|---|---|---|---|---|
| 1 | 10 | 50 | 950 | 0.5 |
| 2 | 0.5 | 500 | 500 | 0.25 |
| 3 | 0.25 | 500 | 500 | 0.125 |
| 4 | 0.125 | 500 | 500 | 0.0625 |
| 5 | 0.0625 | 500 | 500 | 0.03125 |
| 6 | 0.03125 | 250 | 750 | 0.0078125 |
| 7 | 0.0078125 | 250 | 750 | 0.001953125 |
In the following experiment, each standard tube requires 350 μL standard solution. Do not directly measure absorbance at this dilution step.
4. Measurement Procedure
| Component | Soil-Free Tube | Blank Tube 1 | Control Tube | Measurement Tube | Standard Tube | Blank Tube 2 |
|---|---|---|---|---|---|---|
| Air-dried soil sample (g) | - | - | 0.1 | 0.1 | - | - |
| Distilled water (μL) | - | 200 | 200 | - | - | - |
| Reagent I (μL) | 200 | - | - | 200 | - | - |
| Reagent II (μL) | 200 | 200 | 200 | 200 | - | - |
| Mix thoroughly and react at 25°C for 1 h. | ||||||
| Reagent III (μL) | 200 | 200 | 200 | 200 | - | - |
| Shake thoroughly for 30 s. Centrifuge at 10000 rpm and 4°C for 10 min. | ||||||
| Supernatant (μL) | 350 | 350 | 350 | 350 | - | - |
| Standard (μL) | - | - | - | - | 350 | - |
| Distilled water (μL) | - | - | - | - | - | 350 |
| Working solution (μL) | 700 | 700 | 700 | 700 | 700 | 700 |
Mix thoroughly. Pipette 1 mL reaction solution into a 1 mL glass cuvette and measure absorbance at 540 nm. Record the readings as Ano soil, Ablank 1, Acontrol, Ameasurement, Astandard, and Ablank 2.
Calculate: ΔAmeasurement= (Asoil-free- Ablank 1) - (Ameasurement- Acontrol)
Calculate: ΔAstandard= Astandard- Ablank 2
The soil-free tube, blank tube 1, and standard curve only need to be performed 1-2 times.
5. Calculation
5.1 Standard Curve
Use the standard tube concentration (X, μmol/mL) and absorbance (Y, ΔAstandard) to establish the standard curve. Substitute ΔA (Y, ΔAmeasurement) into the standard curve formula to calculate the sample concentration (X, μmol/mL).
5.2 S-NiR Activity
Definition of enzyme activity unit: the amount of enzyme required for each g soil sample to reduce 1 μmol NO2-per day is one enzyme activity unit.
S-NiR (U/g) = X × Vreaction÷ W ÷ T = 14.4 × X ÷ W
- Vreaction: total volume of enzymatic reaction, 0.6 mL
- T: reaction time, 1 h = 1/24 d
- W: sample mass, g
Precautions
- Before the formal assay, select 2-3 samples with large expected differences for a pre-experiment. This 50T kit can test 24 samples.
- Required instruments and supplies to be prepared by the user: balance, visible spectrophotometer, 1 mL glass cuvette, adjustable pipette, benchtop centrifuge, oven, 30-50 mesh sieve, constant-temperature water bath or incubator, and distilled water.
- If ΔAmeasurement> 2.5, dilute the sample before measurement and modify the calculation formula accordingly.
- The linear detection range of this kit is 0.001953125-0.5 μmol/mL.
Appendix
The standard curve should be prepared by the customer for greater accuracy. Refer to the operating table above for the steps. The customer may use the standard curve formula or use the absorbance values of each standard well obtained according to the operating table to prepare a standard curve with R2≥ 0.99, then obtain the calculation formula for sample calculation.
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