Soil Neutral Protease Activity Assay Kit
Spectrophotometry
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Soil Neutral Protease Activity Assay Kit - Spectrophotometric Method
Product Code
67032
Product Introduction
Soil protease participates in the transformation of amino acids, proteins, and other protein-containing nitrogenous organic compounds in soil. Its hydrolysates are one of the nitrogen sources for higher plants.
Soil neutral protease activity (S-NPT) catalyzes protein hydrolysis in a neutral environment and is related to soil organic matter content, nitrogen, and other soil properties. Under neutral conditions, S-NPT hydrolyzes casein to produce tyrosine. Under alkaline conditions, tyrosine reduces phosphomolybdic acid compounds to form tungsten blue, which has a characteristic absorption peak at 680 nm.
Package List
| Specification | Item Code | Component | Quantity |
|---|---|---|---|
| 50T | 67032.1 | Reagent One | 1 bottle |
| 50T | 67032.2 | Reagent Two | 1 bottle |
| 50T | 67032.3 | Reagent Three | 1 bottle |
| 50T | 67032.4 | Reagent Four | 1 bottle |
| 50T | 67032.5 | Reagent Five | 1 bottle |
| 50T | 67032.6 | Reagent Six | 1 bottle |
| 50T | 67032.7 | Reagent Seven | 1 bottle |
| 50T | 67032.m | Manual | 1 copy |
Quality Standards and Safety Information
| Raw Material or Packaging Name | Quality Standard | Main Toxicity |
|---|---|---|
| Reagent One | Not specified | Not specified |
| Reagent Two | Not specified | Not specified |
| Reagent Three | Not specified | Not specified |
| Reagent Four | Not specified | Not specified |
| Reagent Five | Not specified | Not specified |
| Reagent Six | Not specified | Not specified |
| Reagent Seven | Not specified | Not specified |
Shipping and Storage
| Shipping | Shipped with ice packs. |
|---|---|
| Storage | Store at 2-8°C, protected from light. |
| Shelf Life | 180 days. |
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 II: Before use, add 6 mL distilled water and dissolve completely. Store unused reagent at 4°C.
- Reagent III: Before use, add 2 mL Reagent VII and heat in a boiling water bath to dissolve. Then add 8 mL Reagent I and mix thoroughly for later use. Store unused reagent at 4°C.
- Reagent IV: Before use, add 40 mL distilled water and dissolve completely. Store unused reagent at 4°C.
- Reagent VI: 0.05 mg/mL standard tyrosine solution. Store at 4°C.
3. Assay Procedure
- Preheat the spectrophotometer for more than 30 minutes.
- Set the wavelength to 680 nm.
- Zero the instrument with distilled water.
3.1 Enzymatic Reaction
| Component or Step | Assay Tube | Control Tube |
|---|---|---|
| Air-dried soil sample | 0.04 g | 0.04 g |
| Reagent I | 300 μL | - |
| Reagent III | - | 300 μL |
| Incubation | Mix thoroughly. Incubate in a 40°C water bath for 30 minutes. Shake 5-6 times to allow the soil sample to fully contact the reaction solution. | |
| Reagent II | 100 μL | 100 μL |
| Centrifugation | Mix thoroughly. Centrifuge at 8000g and 25°C for 10 minutes, then collect the supernatant. | |
3.2 Color Development and Measurement
| Component or Step | Assay Tube | Control Tube | Standard Tube |
|---|---|---|---|
| Supernatant | 150 μL | 150 μL | - |
| Reagent VI | - | - | 150 μL |
| Reagent IV | 700 μL | 700 μL | 700 μL |
| Reagent V | 150 μL | 150 μL | 150 μL |
| Incubation and centrifugation | Mix well. Incubate in a 40°C water bath for 20 minutes. Centrifuge at 8000g and 25°C for 10 minutes. | ||
| Measurement | Take 1 mL of supernatant and read the absorbance of each tube at 680 nm. | ||
The standard tube only needs to be measured once. Prepare one control tube for each assay tube.
Calculation
Unit definition: The amount of enzyme activity that produces 1 mg tyrosine per day from each g of soil sample is defined as one S-NPT activity unit.
S-NPT (mg/d/g soil sample) = C standard × (A assay tube - A control tube) ÷ A standard tube × V total reaction ÷ W ÷ T = 24 × (A assay tube - A control tube) ÷ A standard tube
| Symbol | Meaning | Value |
|---|---|---|
| C standard tube | Standard tube concentration | 0.05 mg/mL |
| V total reaction | Total volume of the reaction system | 0.4 mL |
| T | Reaction time | 30 min = 1/48 d |
| W | Sample mass | 0.04 g |
Precautions
- Before the formal assay, select 2-3 samples with large expected differences for a preliminary test.
- Instruments and supplies required but not provided: visible spectrophotometer, water bath, magnetic stirrer, adjustable pipette, 1 mL glass cuvette, and distilled water.
- This 50T kit can test 24 samples.
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