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.
キットのコンポーネント
- Inducer Stock Solution: 50 mL× 1 ボトル
- 抽出液: 30 mL× 1 ボトル
- 試薬 1: 12 mL× 1 ボトル
- 試薬 2: パウダー× 2 バイアル
- 試薬 3: 15 mL× 1 ボトル
- 試薬 4: 15 mL× 1 ボトル
- 標準: 1 mL× 1 vial
溶液の準備
- Inducer Solution: Dilute the inducer stock solution 10-fold with distilled water before use. 取る 10 mL of inducer stock solution and add 90 蒸留水 mL. 十分に混ぜ合わせてください. Prepare fresh before each use.
- 試薬 2: 追加 1 蒸留水 mL. Aliquot and store at -20°C. 保管できるのは、 2 weeks at -20°C. ご使用の前に, dilute Reagent Two 50-fold with distilled water. 取る 10 μL of Reagent Two and add 490 蒸留水 μL. よく混ぜます.
- 標準: 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.
ノート
- 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
私. サンプル処理
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 時間. Remove samples, dry them with filter paper, and freeze at -20°C for 30 分. 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) の 1:5 に 10. Grind in an ice bath, で遠心分離する 8000 g, 4℃ 10 分, 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. 追加 1 mL of extraction solution per 5 数百万の細胞または細菌. Sonicate the bacteria or cells (力 200 W, sonication 3 秒, 間隔 10 秒, 繰り返す 30 回). で遠心分離します。 8000 g, 4℃ 10 分, collect the supernatant and keep it on ice for testing.
Ⅱ. Assay Steps
- Preheat the visible spectrophotometer for at least 30 分, 波長を調整して 540 nm, 蒸留水でゼロにする.
- Sample Assay:
| 試薬名 | 試験管 | コントロールチューブ | 標準チューブ | ブランクチューブ |
|---|---|---|---|---|
| サンプル | 100 μL | – | – | – |
| 0.1 μmol/mL Standard Solution | – | – | 100 μL | – |
| Distilled Water | – | 375 μL | – | 475 μL |
| 試薬 1 | 375 μL | – | 375 μL | – |
| 試薬 2 | 125 μL | 125 μL | 125 μL | 125 μL |
| 試薬 3 | 250 μL | 250 μL | 250 |
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