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Plant Nitrate Reductase (NR) ELISA Kit Instruction Manual

 Last Update: 2022-08-30
 Source: Internet
 Author: User

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Plant nitrate reductase (NR) ELISA kit instructions

This kit is for research use only
.

Detection range: 96T 6 IU/L -160 IU/L

Purpose of use:
This kit is used to determine the activity of nitrate reductase (NR) in seaweed tissue, cell supernatant and related liquid samples
.

Experimental principle
This kit uses the double antibody sandwich method to determine the level of plant nitrate reductase (NR) in the specimen
.
Coat the microplate with purified plant nitrate reductase (NR) antibody to make a solid-phase antibody, add nitrate reductase (NR) to the microwells coated with monoclonal antibody in turn, and then combine with HRP-labeled nitrate reductase (NR).
) The antibody binds to form an antibody-antigen-enzyme-labeled antibody complex.
After thorough washing, the substrate TMB is added for color development
.
TMB is converted into blue under the catalysis of HRP enzyme and into the final yellow under the action of acid
.
The shade of color is positively correlated with nitrate reductase (NR) in the sample
.
The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of plant nitrate reductase (NR) activity in the samples was calculated from the standard curve
.

composition

1	30x concentrated washing solution	20ml×1 bottle	7	Stop solution	6ml×1 bottle
2	enzyme labeling reagent	6ml×1 bottle	8	Standard (320 IU/L)	0. 5ml×1 bottle
3	ELISA coated plate	12 holes × 8 strips	9	Standard Diluent	1. 5ml×1 bottle
4	Sample Diluent	6ml×1 bottle	10	manual	1 serving
5	Color developer liquid A	6ml×1 bottle	11	Sealing film	2 sheets
6	Color developer liquid B	6ml×1/bottle	12	sealed bag	1

Specimen requirements
1 .
The extraction should be carried out as soon as possible after the specimen collection, and the extraction should be carried out according to the relevant literature
.
If the test cannot be carried out immediately, the specimen can be stored at -20℃, but repeated freezing and thawing should be avoided
.
2．Samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity
.

Steps

Dilution of standard: This kit provides a standard of original times, and the user can dilute it in a small test tube according to the following chart
.

160IU/L	Standard No. 5	Add 150μl of standard dilution to 150μl of double standard
80IU/L	Standard No. 4	150μl of standard No. 5 added to 150μl of standard dilution
40IU/L	Standard No. 3	Add 150μl of standard No. 4 to 150μl of standard dilution
20IU/L	Standard No. 2	Add 150μl of standard No. 3 to 150μl of standard dilution
10IU/L	Standard No. 1	Add 150μl of standard No. 2 to 150μl of standard dilution

Add sample: set up blank wells (the blank control wells do not add sample and enzyme labeling reagents, and the other steps are the same), standard wells, and sample wells to be tested
.
Accurately add 50μl of standard to the ELISA-coated plate, add 40μl of sample diluent to the well of the sample to be tested, and then add 10μl of the sample to be tested (the final dilution of the sample is 5 times)
.
Add the sample to the bottom of the well of the microtiter plate, try not to touch the wall of the well, and shake gently to mix
.
Incubation: Seal the plate with a sealing film and incubate at 37°C for 30 minutes
.
Dosing: Dilute the 30-fold concentrated washing solution with distilled water 30-fold for later use
Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let stand for 30 seconds, and then discard, repeat this process 5 times, and pat dry
.
Add enzyme: Add 50μl of enzyme labeling reagent to each well, except for blank wells
.
Incubation: operation is the same as 3
.
Washing: the operation is the same as 5
.
Color development: first add 50 μl of color developer A to each well, then add 50 μl of color developer B, gently shake and mix, and develop color at 37°C for 10 minutes in the dark.
Termination: Add 50 μl of stop solution to each well to stop the reaction (the blue turns to yellow at this time)
.
Determination: Zero the blank well, and measure the absorbance (OD value) of each well in sequence at a wavelength of 450 nm
.
The measurement should be carried out within 15 minutes after adding the stop solution
.

Summary of operation procedures:

Calculate
the concentration of the standard substance as the abscissa and the OD value as the ordinate, draw a standard curve on the graph paper, and find out the corresponding concentration from the standard curve according to the OD value of the sample; then multiply by the dilution factor; or Use the concentration and OD value of the standard to calculate the linear regression equation of the standard curve, substitute the OD value of the sample into the equation, calculate the sample concentration, and multiply it by the dilution factor, which is the actual concentration of the sample
.

Notes
1.
The kit should be equilibrated at room temperature for 15-30 minutes before use after taking it out of the refrigerated environment.
If the ELISA-coated plate is not used up after opening, the slats should be stored in a sealed bag
.

2.
Concentrated washing solution may crystallize out.
When diluted, it can be heated in a water bath to help dissolve, and the result will not be affected during washing
.

3.
A sampler should be used for each step of sample addition, and its accuracy should be checked frequently to avoid experimental errors
.
It is best to control the sample addition time within 5 minutes.
If the number of samples is large, it is recommended to use a platoon gun to add samples
.

Please make a standard curve at the same time of each measurement, preferably a duplicate hole
.
If the content of the substance to be tested in the sample is too high (the OD value of the sample is greater than the OD value of the first hole of the standard well), please use the sample diluent to dilute a certain number of times (n times) before the determination.
When calculating, please multiply the total dilution at the end.
multiples (×n×5)
.
The sealing film is for single use only to avoid cross-contamination
.

6.
Keep the substrate away from light
.

7.
Strictly follow the instructions in the instructions, and the test result must be determined by the reading of the microplate reader.

8.
All samples, washing solutions and various wastes should be treated as infectious agents
.

9.
Components of different batches of this reagent should not be mixed
.

Storage conditions and validity period
1.
Kit storage: ; 2-8 ℃
.

2.
Validity: 6 months

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