Mouse T cell activation connexin (LAT) kit instruction manual
This article is provided by: Qiao Yu biological professional and technical staff! This kit is for research use only.
Detection range: 48T 25 ng/L -800 ng/L
purpose of usage:
This kit is used to determine the T cell activation-linked protein (LAT) content in mouse serum, plasma and related liquid samples.
Experimental principle
The kit uses a double antibody sandwich assay to determine mouse T cell activation connexin (LAT) levels in the specimen. The purified mouse T cell activated connexin (LAT) antibody coated microplate was used to prepare a solid phase antibody, and T cell activation connexin (LAT) was sequentially added to the microcapsule of the coated monoclonal antibody, and then labeled with HRP. The T cell activation connexin (LAT) antibody binds to form an antibody-antigen-enzyme-labeled antibody complex, which is thoroughly washed and then added to the substrate TMB for color development. TMB is converted to blue under the catalysis of HRP enzyme and converted to the final yellow color by the action of an acid. The color depth is positively correlated with T cell activation connexin (LAT) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the mouse T cell activation connexin (LAT) concentration in the sample was calculated from a standard curve.
Kit composition
1 | 20 times concentrated washing solution | 20ml × 1 bottle | 7 | Stop solution | 3ml × 1 bottle |
2 | Enzyme standard reagent | 3ml × 1 bottle | 8 | Standard product (1600ng/L) | 0.5ml × 1 bottle |
3 | Enzyme label coated plate | 12 holes × 4 | 9 | Standard dilution | 1.5ml × 1 bottle |
4 | Sample diluent | 3ml × 1 bottle | 10 | Instruction manual | 1 copy |
5 | Developer A solution | 3ml × 1 bottle | 11 | Sealing film | 2 sheets |
6 | Developer B solution | 3ml×1/bottle | 12 | sealed bag | 1 |
Specimen requirements
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 the test cannot be performed immediately, the specimen can be stored at -20 °C, but repeated freezing and thawing should be avoided.
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 a small tube according to the following chart.
800 ng/L | Standard No. 5 | Add 150 μl of standard dilution to 150 μl of the original standard |
400 ng/L | Standard No. 4 | Add 150 μl of standard dilution to 150 μl of standard #5 |
200 ng/L l | Standard No. 3 | Add 150 μl of standard dilution to 150 μl of standard #4 |
100 ng/L | Standard 2 | Add 150 μl of standard dilution to 150 μl of Standard #3 |
50 ng/L | Standard No. 1 | 150 μl of Standard 2 is added to 150 μl of standard dilution |
2. Adding samples: set blank holes respectively (the blank control wells are not added with the sample and the enzyme standard reagent, the other steps are the same), the standard holes, and the sample holes to be tested. Accurately load 50 μl of the standard on the enzyme-labeled plate, add 40 μl of the sample dilution to the well to be tested, and 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 microplate, try not to touch the wall of the well, and shake gently to mix.
3. Incubation: The plate was sealed with a sealing film and incubated at 37 ° C for 30 minutes.
4. Liquor: 20 times concentrated washing solution diluted with distilled water 20 times and used
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 50 μl of color developer A, and then add 50 μl of color developer B, gently shake and mix, and color for 15 minutes at 37 °C.
10. Termination: 50 μl of stop solution was added to each well to terminate the reaction (when 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. The measurement should be carried out within 15 minutes after the addition of the stop solution.
Precautions:
1. The kit should be taken out from the refrigerated environment and should be used at room temperature for 15-30 minutes before use. If the enzyme label is unsealed after opening, the slats should be stored in a sealed bag.
2. Concentrated washing liquid may crystallize out. When diluted, it can be heated and dissolved in a water bath. The washing will not affect the result.
3. The sampler should be used for each step, and the accuracy should be corrected frequently to avoid test errors. The loading time is controlled within 5 minutes. If the number of specimens is large, it is recommended to use a gun.
4. Please make a standard curve at the same time for each measurement and make a double hole. If the content of the substance to be tested in the specimen is too high (the OD value of the sample is larger than the OD value of the standard pore), please first dilute the sample dilution with a certain multiple (n times) and then measure it. When calculating, multiply the total dilution factor ( ×n×5).
5. The sealing film is intended for single use only to avoid cross-contamination.
6. Please keep the substrate away from light.
7. Strictly follow the instructions of the manual, the test results must be based on the microplate reader reading.
8. All samples, washings and various wastes should be treated as infectious materials.
9. The different batch components of this reagent must not be mixed.
10. In the case of an English manual, the English manual shall prevail.
Storage conditions and expiration date
1. The kit is stored at: 2-8 °C.
2. Validity: 6 months
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