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Rat Prothrombin Kit Instructions for Use
The kit uses a double antibody sandwich method to determine the level of rat prothrombin in the specimen. Larger purified
The prothrombin antibody is coated with a microplate to prepare a solid phase antibody, which is sequentially added to the microcapsule of the coated monoclonal antibody.
Prothrombin is then combined with HRP-labeled prothrombin antibody to form an antibody-antigen-enzyme-labeled antibody complex, which is thoroughly washed and then plated with TMB. TMB is converted to blue under the catalysis of HRP enzyme.
1. The specimens should be extracted as soon as possible after collection, and the extraction should be carried out according to the relevant literature. The experiment should be carried out as soon as possible after extraction. If not
Immediately test, the specimen can be stored at -20 ° C, but should avoid repeated freezing and thawing
2. Samples containing NaN3 could not be detected because NaN3 inhibited horseradish peroxidase (HRP) activity.
Steps
1. Dilution of standard: This kit provides one original standard, which can be diluted in small tubes according to the following chart.
release
2. Adding samples: set blank holes separately (the blank control holes are not added with samples and enzyme labeling reagents, the other steps are the same), standard holes,
Sample hole to be tested. Accurately load 50 μl of the standard on the enzyme-labeled plate, and add 40 μl of the sample dilution to the sample well.
Then add 10 μl 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 ELISA plate.
Do not touch the wall of the hole and mix gently by shaking.
3. Incubation: After sealing with a sealing film, incubate at 37 ° C for 30 minutes.
4. Dosing: Dilute 30 times concentrated washing solution with distilled water 30 times.
5. Washing: Carefully remove the sealing film, discard the liquid, dry it, fill each well with the washing solution, let stand for 30 seconds, then discard it.
Repeat 5 times and pat dry.
6. Add enzyme: Add 50 μl of enzyme labeling reagent to each well, except for blank wells.
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 to each well, then add 50μl of color developer B, gently shake and mix, avoid color at 37 °C
15 minutes.
10. Termination: 50 μl of stop solution was added to each well to stop the reaction (the blue color turned yellow).
11. Measurement: The absorbance (OD value) of each well was measured sequentially with a blank air conditioner of zero and a wavelength of 450 nm. Determination should be terminated
It can be carried out within 15 minutes after the liquid.