Human MEK kinase enzyme-linked immunoassay
Kit instruction manual
Read this manual carefully before use. This ELISA kit is based on the principle of double antibody sandwich technology to detect human MEK kinase, which can only be used for research purposes, not for medical diagnosis.
Uses: Used for the determination of MEK kinase in human serum, plasma and related liquid samples.
working principle
This kit uses a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) to determine the level of human MEK kinase in the sample. Add MEK kinase to the enzyme-labeled wells precoated with human MEK kinase monoclonal antibody and incubate; after washing, add HRP-labeled MEK kinase antibody. After incubation and washing, the unbound enzyme is removed, and then the substrates A and B are added to produce a blue color, which is converted into a final yellow color under the action of an acid. The color depth is positively correlated with the concentration of human MEK kinase in the sample.
Kit composition
1
Standard product (480pmol / L)
0.5ml
7
Developer A liquid
3ml
2
Standard dilution
3ml
8
Developer B liquid
3ml
3
Enzyme coated plate
12 holes × 4 strips
9
Stop solution
3ml
4
Enzyme reagent
3ml
10
Instructions
1 serving
5
20 times concentrated washing liquid
20ml
11
Sealing film
2 sheets
6
Sample diluent
3ml
12
sealed bag
1
Reagents and equipment needed but not provided
1. 37 ℃ thermostat.
2. Standard specification microplate reader.
3. Precision pipettes and disposable tips
4. Distilled water,
5. Disposable test tube
6. Absorbent paper
Precautions
1. The kit taken from 2-8 ° C should be equilibrated at room temperature for at least 30 minutes before opening the kit. If the enzyme-coated plates are not used up after opening, the slats should be stored in sealed bags.
2. The sampler should be used at each step of sample addition, and the accuracy should be regularly checked to avoid experimental errors.
3. It is recommended that all standards and samples be tested in duplicate. If the content of the substance to be tested in the specimen is too high, please dilute it with a certain multiple of the sample diluent (n times) and then perform the measurement according to the instructions. When calculating, please multiply the total dilution factor (× n × 5).
4. Strictly follow the instructions, and the test results must be determined by the microplate reader.
5. To avoid cross-contamination, avoid repeated use of the tip and sealing film in your hand.
6. Unused other reagents should be packed or covered. Do not mix reagents of different batches. Use before warranty.
7. Substrate B is sensitive to light and avoid prolonged exposure to light.
Washing method
Manual plate washing method: throw away the liquid in the enzyme label plate; place a few layers of absorbent paper on the experimental table, the enzyme label plate is forced to pat several times downward; inject at least 0.35ml of the diluted washing solution into the well and soak 2 minutes. Repeat this process several times as needed.
Automatic plate washing: If there is an automatic plate washing machine, it should be used in the formal experiment process after being used proficiently
Specimen requirements
1. The samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity.
2. Specimens are extracted as soon as possible after collection, and extraction is performed according to relevant literature, and experiments should be conducted as soon as possible after extraction. If the test cannot be performed immediately, the specimen can be stored at -20 ℃, but repeated freezing and thawing should be avoided
Operating procedures
1. Dilution of standard products (this kit provides one original standard, please follow the instructions to dilute it in a small test tube):
240pmol / L
(No. 5 standard product)
120μl of original standard is added to 120μl of standard dilution
120pmol / L
(Standard 4)
120μl No. 5 standard is added to 120μl standard dilution
60 pmol/L
(Standard No. 3)
120μl No. 4 standard is added to 120μl standard dilution
30 pmol/L
(Standard No. 2)
Add 120μl of standard No. 3 to 120μl of standard dilution
15 pmol/L
(No. 1 standard product)
120μl No. 2 standard is added to 120μl standard dilution
2. Separately set up blank wells (the blank control wells do not add samples and enzyme label reagents, the rest of the steps are the same), standard wells, sample wells to be tested. Add 50μl of diluted standard to the standard wells of the enzyme-coated plate; add 40μl of the sample dilution to the sample wells of the enzyme-coated plate and then add 10μl of the sample to be tested (the final dilution of the sample is 5 Times). Shake gently to mix, and incubate at 37 ° C for 30 minutes.
3. Discard the liquid and spin dry. Fill each well with 30-fold diluted washing liquid, shake for 30 seconds, shake off the washing liquid, and pat dry with absorbent paper. Repeat this 5 times, pat dry.
4. Add 50μl of enzyme-labeled reagent to each well, except for blank wells. Shake gently to mix, and incubate at 37 ° C for 30 minutes.
5. Discard the liquid and spin dry. Fill each well with 30-fold diluted washing liquid, shake for 30 seconds, shake off the washing liquid, and pat dry with absorbent paper. Repeat this 5 times, pat dry.
6. Add 50μl of developer A to each well, then add 50μl of developer B, mix gently, and develop at 37 ° C in the dark for 10 minutes.
7. Remove the enzyme labeling plate, add 50μl of stop solution to each well to stop the reaction (at this time, the blue will turn to yellow).
8. Determination: Zero the blank holes, and measure the absorbance (OD value) of each well at 450nm wavelength. The measurement should be carried out within 15 minutes after adding the stop solution.
9. The linear regression equation of the standard curve is calculated according to the concentration of the standard product and the corresponding OD value, and then the corresponding sample concentration is calculated on the regression equation according to the OD value of the sample. It can also be calculated using various application software. It should be remembered that since the sample is diluted, its actual concentration should be multiplied by the total dilution factor.
Summary of operating procedures:
Prepare reagents, samples and standards
Add prepared samples and standards, and react at 37 ℃ for 30 minutes
Wash the plate 5 times, add enzyme reagent and react at 37 ℃ for 30 minutes
Wash the plate 5 times, add color developing solutions A and B, and develop at 37 ℃ for 10 minutes
Add stop solution
Read OD value within 15 minutes
Calculation
Detection range: 10 pmol/L→300pmol/L.
Specification: 48T / box
Storage: 2-8 ℃
Validity: 6 months (2-8 ℃).
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