Soil Nitrite Reductase(S-NiR) Activity Assay Kit
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Soil Nitrite Reductase Activity Assay Kit - Microplate Method
Product Code
67054
Product Introduction
Soil nitrite reductase is one of the key enzymes in denitrification. It participates in the reduction of nitrite to NO, and its activity reflects nitrogen conversion efficiency during biodegradation. This assay provides a basis for studying nitrogen transformation patterns in soil.
Nitrite reductase reduces NO2-to NO, decreasing the amount of NO2-available for the diazotization reaction that forms a purplish-red compound. The change in absorbance at 540 nm reflects soil nitrite reductase activity.
Example Measurement Data
Sample: forest soil.
| Measurement | OD540 nm |
|---|---|
| No soil | 0.915 |
| Blank 1 | 0.051 |
| Control | 0.082 |
| Assay | 0.774 / 0.779 |
Actual readings may vary depending on the detection instrument and conditions. The data above are for reference only.
Package Contents and Storage
Kit size: 100T.
| Code | Component | Quantity | Storage |
|---|---|---|---|
| 67054.1 | Reagent I | 2.5 mL | 2-8°C, protected from light |
| 67054.2 | Reagent II | 0.05 g | 2-8°C |
| 67054.3 | Reagent III | 5 mL | 2-8°C |
| 67054.4 | Reagent IV | 8 mL | 2-8°C, protected from light |
| 67054.5 | Reagent V | 8 mL | 2-8°C, protected from light |
| 67054.6 | Standard | 1 mL | 2-8°C, protected from light |
| 67054.m | Manual | 1 copy | - |
Quality and Safety Information
| Material | Quality Standard | Main Toxicity |
|---|---|---|
| Reagent I | - | - |
| Reagent II | - | - |
| Reagent III | - | - |
| Reagent IV | - | - |
| Reagent V | - | - |
| Standard | - | - |
Transportation and Storage
Transportation: this product is transported with ice packs.
Storage: store each component according to the instructions above. Shelf life: 180 days.
Instructions for Use
1. Sample Processing
Naturally air-dry fresh soil samples or dry them in a 37°C oven, then pass them through a 30-50 mesh sieve.
2. Reagent Preparation
- Reagent II: before use, add 5 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 return to 4°C or -20°C storage.
- Reagent III: if precipitation occurs, heat at 60-80°C to dissolve.
- Reagent IV: if precipitation occurs, heat at 60-80°C to dissolve.
- Standard: 10 µmol/mL sodium nitrite.
- Working solution: before use, mix Reagent IV and Reagent V at a 1:1 ratio according to the required amount. Prepare fresh before use. After preparation, it is valid for 48 h at 4°C.
3. Operating Procedure
- Preheat the microplate reader for at least 30 min. Set the wavelength to 540 nm and zero with distilled water.
- Dilute the 10 µmol/mL standard solution with distilled water to prepare 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.0078125, and 0.001953125 µmol/mL standard solutions.
Standard Solution Dilution
| No. | Concentration Before Dilution (µmol/mL) | Standard Solution Volume (µL) | Distilled Water Volume (µL) | Concentration After Dilution (µmol/mL) |
|---|---|---|---|---|
| 1 | 10 | 40 | 360 | 1 |
| 2 | 1 | 200 | 200 | 0.5 |
| 3 | 0.5 | 200 | 200 | 0.25 |
| 4 | 0.25 | 200 | 200 | 0.125 |
| 5 | 0.125 | 200 | 200 | 0.0625 |
| 6 | 0.0625 | 200 | 200 | 0.03125 |
| 7 | 0.03125 | 100 | 300 | 0.0078125 |
| 8 | 0.0078125 | 100 | 300 | 0.001953125 |
In the following experiment, each standard tube requires 70 µL standard solution. Do not measure absorbance directly 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) | - | - | 0.02 | 0.02 | - | - |
| Distilled water (µL) | - | 40 | 40 | - | - | - |
| Reagent I (µL) | 40 | - | - | 40 | - | - |
| Reagent II (µL) | 40 | 40 | 40 | 40 | - | - |
| Mix thoroughly, then react at 25°C for 1 h. | ||||||
| Reagent III (µL) | 40 | 40 | 40 | 40 | - | - |
| Shake thoroughly for 30 s, then centrifuge at 10,000 rpm and 4°C for 10 min. | ||||||
| Supernatant (µL) | 70 | 70 | 70 | 70 | - | - |
| Standard (µL) | - | - | - | - | 70 | - |
| Distilled water (µL) | - | - | - | - | - | 70 |
| Working solution (µL) | 140 | 140 | 140 | 140 | 140 | 140 |
Mix thoroughly, then transfer 200 µL reaction solution to a 96-well plate. Measure absorbance at 540 nm and record the values as Asoil-free, 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 run 1-2 times.
Calculation
1. Standard Curve Preparation
Use the standard tube concentration (X, µmol/mL) and absorbance (Y, ΔAstandard) to establish the standard curve. Substitute ΔAmeasurementas Y into the standard curve equation to calculate the sample concentration X (µmol/mL).
2. S-NiR Activity Calculation
Definition of enzyme activity unit: the amount of enzyme required to reduce 1 µmol NO2-per day per gram of soil sample is one enzyme activity unit.
S-NiR (U/g) = X × Vreaction÷ W ÷ T = 2.88 × X ÷ W
- X: sample concentration calculated from the standard curve, µmol/mL
- Vreaction: total enzymatic reaction volume, 0.12 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 preliminary test. This 100T kit can test 48 samples.
- Required instruments and supplies: balance, microplate reader, 96-well plate, adjustable pipette, desktop 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 0.001953125-1 µmol/mL.
Appendix
The standard curve is more accurate when prepared by the customer. Follow the measurement procedure above. Use either the standard curve formula or the absorbance values from each standard well to prepare a standard curve with R2≥ 0.99, then use the resulting equation for sample calculation.
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