Product Overview
NR (EC 1.7.1.3) is a widely found enzyme in plants. It plays a crucial role in converting nitrate nitrogen to ammonia nitrogen and is also an inducible enzyme, affecting crop yield and quality. NR catalyzes the reduction of nitrate to nitrite: NO3- + NADH+H+ → NO2- + NAD+ + H2O. Under acidic conditions, the produced NO2- can participate in a diazotization reaction to form a magenta-colored compound. This compound has an absorption peak at 540 nm, and the change in absorbance at 540 nm can be used to represent enzyme activity.
Kit Components
- Inducer Stock Solution: 50 mL x 1 bottle
- Extraction Solution: 30 mL x 1 bottle
- Reagent One: 12 mL x 1 bottle
- Reagent Two: Powder x 2 vials
- Reagent Three: 15 mL x 1 bottle
- Reagent Four: 15 mL x 1 bottle
- Standard: 1 mL x 1 vial
Solution Preparation
- Inducer Solution: Dilute the inducer stock solution 10-fold with distilled water before use. Take 10 mL of inducer stock solution and add 90 mL of distilled water. Mix thoroughly. Prepare fresh before each use.
- Reagent Two: Add 1 mL of distilled water. Aliquot and store at -20°C. It can be stored for 2 weeks at -20°C. Before use, dilute Reagent Two 50-fold with distilled water. Take 10 μL of Reagent Two and add 490 μL of distilled water. Mix well.
- Standard: Prepare a 0.1 μmol/mL sodium nitrite standard solution by diluting the 10 μmol/mL sodium nitrite standard solution 100-fold with distilled water before use.
Notes
- If the absorbance is greater than 0.8, dilute the sample with extraction solution. Pay attention to adjusting the dilution factor accordingly in the calculation formula.
- Strictly follow the order of adding reagents listed in the sample assay table for the experiment.
Experimental Procedures
I. Sample Treatment
Tissue Pre-treatment:
- Add an appropriate amount of inducer solution to a beaker. Wash fresh samples, dry them with filter paper, and place them in the inducer solution (enough to submerge). Incubate in the dark for 2 hours. Remove samples, dry them with filter paper, and freeze at -20°C for 30 minutes. Thaw samples and dry them with filter paper again. (Perform induction treatment as needed. Generally, induction treatment is not required. If the pre-experiment results show no activity, induction treatment is necessary.)
- Weigh approximately 0.1 g of sample and add 1 mL of extraction solution according to the ratio of tissue weight (g) to extraction solution volume (mL) of 1:5 to 10. Grind in an ice bath, centrifuge at 8000 g, 4°C for 10 minutes, and collect the supernatant. Keep the supernatant on ice for testing.
Cell or Bacteria Pre-treatment:
- Collect cell or bacteria samples in a centrifuge tube and discard the supernatant. Add 1 mL of extraction solution per 5 million cells or bacteria. Sonicate the bacteria or cells (power 200 W, sonication 3 seconds, interval 10 seconds, repeat 30 times). Centrifuge at 8000 g, 4°C for 10 minutes, collect the supernatant and keep it on ice for testing.
II. Assay Steps
- Preheat the visible spectrophotometer for at least 30 minutes, adjust the wavelength to 540 nm, and zero with distilled water.
- Sample Assay:
| Reagent Name | Test Tube | Control Tube | Standard Tube | Blank Tube |
|---|---|---|---|---|
| Sample | 100 μL | – | – | – |
| 0.1 μmol/mL Standard Solution | – | – | 100 μL | – |
| Distilled Water | – | 375 μL | – | 475 μL |
| Reagent One | 375 μL | – | 375 μL | – |
| Reagent Two | 125 μL | 125 μL | 125 μL | 125 μL |
| Reagent Three | 250 μL | 250 μL | 250 |
Reviews
There are no reviews yet.