Mouse Calcineurin (CaN) Kit Instructions for Use
This kit is used to determine calcineurin (CaN) levels in serum, plasma and related fluid samples in mice.
Experimental principle
The kit uses a double antibody sandwich assay to determine the level of calcineurin (CaN) in mice. Purified mouse
The calcineurin (CaN) antibody is coated with a microplate to prepare a solid phase antibody, and calcium is sequentially added to the microcapsule of the coated monoclonal antibody.
Neurophosphatase (CaN), which binds to HRP-labeled calcineurin (CaN) antibody to form an antibody-antigen-enzyme label
The antibody complex was thoroughly washed and added to the substrate TMB for color development. TMB is converted to blue under the catalysis of HRP enzyme, and
It is converted to the final yellow color by the action of an acid. The color depth is positively correlated with calcineurin (CaN) in the sample.
The absorbance (OD value) was measured at 450 nm using a microplate reader, and the mouse calcineurin was calculated from the standard curve.
Acidase (CaN) concentration.
Kit composition
1 30 times concentrated washing solution
2 enzyme standard reagent
3 enzyme label coating board
4 sample diluent
5 developer A solution
6 developer B liquid
Specimen requirements
20ml × 1 bottle
6ml × 1 bottle
12 holes × 8
6ml × 1 bottle
6ml × 1 bottle
6ml×1/bottle
7 stop solution
8 standard products (48U/L)
9 standard dilution
10 instructions
11 sealing film
12 sealed bag
6ml × 1 bottle
0.5ml × 1 bottle
1.5ml × 1 bottle
1 serving
2 sheets
1
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.
24U/L
12U/L
6U/L
3U/L
1.5U/L
Standard No. 5
Standard No. 4
Standard No. 3
Standard 2
Standard No. 1
Add 150 μl of standard dilution to 150 μl of standard dilution
Add 150 μl of standard dilution to 150 μl of standard #5
Add 150 μl of standard dilution to 150 μl of standard #4
Add 150 μl of standard dilution to 150 μl of standard #3
150 μl of Standard 2 is added to 150 μl of standard dilution
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
10 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.
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