Instruction Manual of Mouse Urine Protein (UP) ELISA Kit
This kit is for research use only.


Drug Name:
Generic Name: Mouse Urine Protein (UP) ELISA Kit

purpose of usage:
This kit is used to determine the content of urine protein (UP) in mouse serum and plasma.

Experimental principle This kit uses the double antibody sandwich method to determine the level of mouse urine protein (UP) in the specimen. With purified anti-urinary protein (UP)

The antibody is coated on the microwell plate to make a solid phase antibody. Urine protein (UP) is added to the monoclonal antibody-coated microwell and then labeled with HRP

UP) The antibody binds to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB is added

Urine protein (

Color rendering. TMB is converted into blue under the catalysis of HRP enzyme, and into the final yellow under the action of acid. Deep color

There is a positive correlation between the shallow and the urine protein (UP) in the sample. Measure the absorbance (OD value) at 450nm with a microplate reader, pass

Calculate the content of mouse urine protein (UP) in the sample through the standard curve.

Kit composition
1 20 times concentrated washing solution 30ml × 1 bottle 7 stop solution 6ml × 1 bottle


2 Enzyme label reagent 6ml × 1 bottle 8 standard (225μmol / L) 0.5ml × 1 bottle


3 Enzyme label coating plate 12 wells × 8 strips 9 Standard dilutions 1.5ml × 1 bottle


4 Sample diluent 6ml × 1 bottle 10 Instructions 1 copy


5 Developer A solution 6ml × 1 bottle 11 sealing film 2 sheets


6 Developer B solution 6ml × 1 / bottle 12 sealed bag 1


Specimen processing and requirements Serum and plasma samples can be directly measured;

2. The sample containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity.

3. Carry out the experiment as soon as possible after collection. If the experiment cannot be performed immediately, the specimen can be stored at -20 ° C.

Refreeze and thaw.

Steps
1. Dilution and sample loading of standard products: set 10 standard wells in sequence on the enzyme-coated plate, and separate them in the first and second wells.

Add 100μl of standard, then add 50μl of standard diluent to the first and second wells, mix well; then take 100μl each

Do not add to the third and fourth wells, and then add 50μl of standard dilution solution to the third and fourth wells respectively.

Take 50μl each of the three and fourth wells and discard; then take 50μl each and add them to the fifth and sixth wells respectively;

Add 50ul of standard dilution solution to the sixth well and mix well; after mixing, take 50μl from the fifth and sixth wells respectively

Go to the seventh and eighth wells; add 50μl of the standard diluent to the seventh and eighth wells respectively.

Take 50μl from the eighth well and add it to the ninth and tenth wells; add 50μl of the standard dilution solution to the ninth and tenth wells,

After mixing, take 50μl from the ninth and tenth wells and discard. (After dilution, the volume of each well is 50μl, and the concentration is 150

μmol / L, 100μmol / L, 50μmol / L, 25μmol / L, 12.5μmol / L).

2. Add sample: set blank wells separately (the blank control wells do not add samples and enzyme reagents, the rest of the steps are the same), the sample to be tested

Pinhole. Add 40μl of sample diluent to the test sample well of the enzyme-coated plate, and then add 10μl of the test sample (sample

The final dilution of the product is 5 times). Add the sample and add the sample to the bottom of the well of the microplate, shake gently to mix. 3. Incubation: seal the plate with the sealing film and incubate at 37 ° C for 30 minutes.

4. Mixing solution: dilute 20 times concentrated washing solution with distilled water 20 times and reserve

5. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing liquid, let stand for 30 seconds and then discard, so

Repeat 5 times and pat dry.

6. Add enzyme: add 50μl of enzyme label reagent to each well, except blank well.

7. Incubation: The operation is the same as 3.

8. Washing: The operation is the same as 5.

9. Color development: add 50μl of developer A to each well, and then add 50μl of developer B, mix gently, and develop color at 37 ℃ in the dark

15 minutes.

10. Termination: Add 50μl of stop solution to each well to stop the reaction (at this time the blue will turn to yellow).

11. Determination: Measure the absorbance (OD value) of each well in sequence with the blank air conditioner at zero and 450nm wavelength. Determination should be terminated

Within 15 minutes after the solution.


Calculation

Taking the concentration of the standard as the abscissa and the OD value as the ordinate, draw a standard curve on the coordinate paper, according to the sample

The corresponding concentration of OD value is found by the standard curve; then multiplied by the dilution factor; or the standard concentration and OD value are used to calculate the standard

The linear regression equation of the quasi-curve, the OD value of the sample is substituted into the equation, the sample concentration is calculated, and then multiplied by the dilution factor,

This is the actual concentration of the sample.

Precautions
1. The kit should be equilibrated at room temperature for 15-30 minutes before being taken out of the refrigerated environment.

After use, the slats should be stored in sealed bags.

2. Crystals may be precipitated in the concentrated washing liquid, which can be heated and dissolved in a water bath during dilution, and the results will not be affected during washing.

3. The sampler should be used at each step of sample addition, and the accuracy should be regularly checked to avoid test errors. One sample loading time is best

Control within 5 minutes, if the number of specimens is large, it is recommended to use a volley gun to add samples.

4. Please make a standard curve at the same time of each measurement, it is best to make a double hole. If the content of the test substance in the specimen is too high (sample OD value

Greater than the OD value of the first well of the standard product), please dilute it with a certain multiple (n times) of the sample diluent before measuring.

When calculating, please multiply the total dilution factor (× n × 5).

5. Strictly follow the instructions, and the test results must be determined by the reading of the microplate reader.

6. The components of different batches of this reagent shall not be mixed.

7. The sealing film is limited to one-time use to avoid cross-contamination.

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

9. Please keep the substrate away from light.

examination range:
5μmol / L -200μmol / L

specification:
96 servings / box

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

2. Validity: 6 months

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