Nitrate Reductase (NR) Activity Assay Kit Instructions

NoteMeaning: Before the official measurement, make sure to take 2-3 samples with large expected differences for the pre-measurement

Measurement meaning:

NR (EC 1.7.1.3) is common in plants and is a plant. key enzyme that converts nitrate nitrogen into ammoniacal nitrogen is also an induced enzyme that affects crop yield and quality.

Determination principle:

 NR catalyzes the reduction of nitrate to nitrite, NO3ˉ +NADH+H+→ NO2ˉ +NAD+++H 2O; the nitrite produced can be formed quantitatively with p-aminobenzene sulfonic acid and α-naphthylamine under acidic conditions Red azo compound; the generated red azo compound has a maximum absorption peak at 540 nm, which can be determined by spectrophotometry.

Must your owntools and to bring >>>Equipments:

Visible spectrophotometer/microplate reader, water bath, benchtop centrifuge, adjustable pipette, microquartz cuvette/96-well plate, mortar, ice and distilled water.

Reagent composition and preparation:<!-- p Inducer stock solution: liquid 50mL×1 bottle, store at 4°C.

Extraction solution: liquid 60mL×1 bottle, store at 4°C.

Reagent 1: Liquid 10mL×1 bottle, store at -20°C.

Reagent 2: liquid 5mL×1 bottle, store at -20°C;

Reagent 3: liquid6mL×1 bottle, store at 4°C (if crystallization occurs, store at 60°C-90°C) ℃ before use after dissolving in a water bath);

Reagent 4: liquid 6mL×1 bottle , store at 4℃.

Reagent 5: Standard stock solution 1 ml, store at -20°C.

Preparation of the inducer application solution: when you use it, dilute the inducer stock solution 10 times, that is, take 10ml of the inducer stock solution and add 90ml of distilled water, and mix well.

Preparation of 0.1umol/ml standard solution: When using, dilute reagent 5 100 times, that is, take 0.1ml of reagent 5 and add 9.9ml of distilled water, and mix well.

Pre-processing example:

Pre-treatment of animal and plant tissue samples:

(1) Take an appropriate amount of inducer in a beaker, wash the fresh sample, dry it with filter paper and put it in the inducer application.ossing (just submerge), soak for 2 hours, remove the sample and pat dry with filter paper.

(2) According to the ratio of tissue mass (g) to extraction volume (ml) of 1:5~10 (it is recommended to weigh about 0.1g of tissue and add 1ml of extract) , feed homogenizing pulp into an ice bath. Centrifuge at 8000 g for 10 minutes at 4°C, take the supernatant and place on ice to test.

Note:

1. It is recommended to use fresh samples that have not been frozen.

2. In general, no induction treatment is required. If there is no activity in the pre-assay result (A-determination ≤ a control tube), induction treatment is required.

Pre-treatment of bacteria or cultured cells:

First collect bacteria or cells in a pennyrifuge tube, discard supernatant after centrifugation; according to the number of bacteria or cells (104 ): The extract volume (ml) is 500~1000:1 (it is recommended to add 1ml extract to 5 million bacteria or cells), disrupt bacteria or cells ultrasonic (ice bath, power 20% or 200W, ultrasonic 3s, interval 10s, repeat 30 times); centrifuge at 8000g 4°C for 10min, take the supernatant and place on ice to test.

DeterminationSteps:

1. Preheat the spectrophotometer or microplate reader for more than 30 minutes, adjust the wavelength to 540 nm, zero the distilled water.

2. Sample determination (add the following reagents to EP tube or 96-well plate)

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