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Nitrate Reductase (NR)Assay Kit/SLBC0085

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Product Detail

Nitrate Reductase (NR)Assay Kit

Note: It is necessary to predict 2-3 large difference samples before the formal determination.

Operation Equipment: Spectrophotometer/ Microplate reader

Catalog Number: SLBC0085

Size:100T/96S

Components:

Inducer reserve fluid: Liquid 100 mL×1. Storage at 4℃. It should be prepared before using with 10 times dilution. Prepare the reagent when it will be used.

Extraction reagent: Liquid 120 mL×1 . Storage at 4℃.

Reagent I: Liquid 15 mL×1. Storage at -20℃.

Reagent II: Powder ×1. Storage at -20℃. Add 2 mL of Extraction reagent before using, mix thoroughly. For long term preservation, separate into small tubules and storage at -20℃, avoid repeated freezing and thawing. It can be stored for two weeks at -20℃.

Product Description

NR (EC 1.7.1.3) is a key enzyme in the transformation of plant nitrate nitrogen into ammonia nitrogen as well as an induction enzyme, widely exists in plants and has an impact on crop yield and quality.

NR catalyzed nitrate reduction to nitrite, with NO₃ˉ + NADH + H^＋→ NO₂ˉ + NAD^＋+ H₂O. NADH has a characteristic absorption peak at 340 nm. The change of absorbance at 340 nm can indicate the enzyme activity.

Reagents and Equipment Required but Not Provided.

Table centrifuge, water-bath, micro quartz cuvette/96 well UV flat-bottom plate, spectrophotometer/ microplate reader, mortar/ homogenate, ice and distilled water.

Procedure

I. Sample Preparation

1. Put proper inducers in a beaker, wash fresh specimens and then drain with filter paper. Put the specimens in the inducer applied liquid (covered), protected from light, immerse 2 hours. Take out the samples and drain with filter paper. Frozen at -20℃ for 30 minutes, then take out the sample, drain with filter paper. (Conduct induction treatment as needed)

2. Put 0.1 g of induced sample into 1 mL of Extraction reagent and fully grinding on ice. Centrifuge at 4000×g for 10 minutes at 4℃ to remove insoluble materials, and take the supernatant on ice before testing.

II. Determination procedures

1. Preheat spectrophotometer/microplate reader for more than 30 minutes, adjust the wavelength to 340 nm, set the counter to zero with distilled water.

2. Sample determination (adding the following reagents to the EP tube)

Reagent (μL)	Test Tube (A_T)	Blank Tube (A_B)
Sample	12	-
Extraction reagent	68	80
Reagent I	108	108
Reagent Ⅱ	12	12

Mix thoroughly, detect the initial value at 340 nm and noted as A_T1, A_B1.Thenreact at 25℃(other species) or 37℃(mammal) for 30 minutes and detect the absorbance at 340 nm and noted as A_T2/A_B2.Calculate the change for the samples and blank control: ΔA_T=A_T1-A_T2, ΔA_B=A_B1-A_B₂, ΔA=ΔA_T-ΔA_B

Note: Blank tubes only need to be tested 1-2 times.

III. Calculation

a. micro quartz cuvette

1. Sample weight:

Unit definition: One unit of enzyme activity is defined as the amount of enzyme catalyzes the consumption of 1 μmol of NADH in the reaction system per hour every gram sample.

NR (U/g fresh weight) =[ΔA×Vt÷(ε×d) ×10⁶ ]÷(W÷Ve×Vs)÷T =5.359×ΔA÷W.

2. Calculate by protein concentration:

Unit definition: One unit of enzyme activity is defined as the amount of enzyme catalyzes the consumption of 1 μmol of NADH in the reaction system per hour every milligram protein.

NR(U/mg prot)=[ΔA×Vt÷(ε×d) ×10⁶ ]÷(Vs×Cpr)÷T =5.359×ΔA÷Cpr.

Vt: Total reaction volume, 0.0002 L;

Vs: Sample volume, 0.012 mL;

Ve: Extraction volume, 1 mL;

T: Reaction time, 0.5 h;

ε: Molar extinction coefficient of NADH: 6220 L/mol/cm;

d: Cuvette light path: 1 cm;

Cpr: Sample protein concentration, mg/mL;

W: Fresh weight of the sample, g;

10⁶: Unit conversion factor, 1 mol = 10⁶ μmol.

b. 96 well UV flat-bottom plate

Change d (1 cm) in the formula to 0.6 cm (light path of 96 well UV plate).

Note:

1. When the measured absorbance value is greater than 1.5 or ΔA greater than 0.5, recommends that dilute supernatant fluid.

2. When measured value of ΔA is too small (less than 0.01), prolong the reaction time(water bath time).

3. It is not recommended to measure too many samples in one measurement to avoid delaying the enzyme reaction for too long.

4. The blank tube is a test hole for testing the quality of each reagent component. Under normal conditions, the change of blank tube shall not exceed 0.05.

Experimental example:

1. Take 0.1 g Buxus microphylla and add 1 mL of Extraction reagent for homogenization, take the supernatant and dilute it twice with Extraction reagent, then operate according to the determination steps, and calculate with micro quartz colorimetric plate: ΔA_T= A_T1- A_T2= 1.4285-1.1089 = 0.3196, ΔA_B= A_B1- A_B2= 0.8794-0.8334 = 0.046, ΔA= ΔA_T- ΔA_B= 0.3196-0.046=0.2736 The enzyme activity was calculated as follows

NR (U/g mass) = 5.359÷ΔA÷W×2(dilution ratio) =5.359×0.2736÷0.1×2=29.3244 U / g mass.

2. Take 0.1 g of Yulan and add 1 mL of Extraction reagent for homogenization, take the supernatant and dilute it twice with the Extraction reagent, then operate according to the determination steps, measure and calculate with micro quartz colorimetric plate: ΔA_T= A_T1- A_T2= 1.4696-1.0602=0.4094, ΔA_B= A_B1- A_B2 = 0.8794-0.8334 = 0.046, ΔA = ΔA_T - ΔA_B= 0.4094-0.046 = 0.3634

NR(U/g mass) = 5.359÷ΔA÷W×2(dilution ratio) = 5.359÷ 0.3634÷0.1×2 = 38.9492 U/g mass.