Mouse phosphorylated AKT protein (p-AKT) detection kit

Mouse

16μmol/L

Each liquid component is clear and transparent, without precipitation or floccules. Microplate aluminum foil bagIt should be vacuum packed without damage or leakage.

Used to detect the concentration of mouse phosphorylated AKT protein (p-AKT) in samples such as serum, plasma, and cell culture supernatant.

This kit uses a double antibody sandwich enzyme-linked immunosorbent assay (ELISA). In a microwell ELISA plate pre-coated with anti-mouse phosphorylated AKT protein (p-AKT) antibody (solid phase antibody), add mouse phosphorylated AKT protein (p-AKT) calibrator and the sample to be tested, then add biotin-labeled antibody, incubate and fully wash, then add HRP-coupled avidin, incubate and fully wash to remove unbound components, and form a sandwich complex of solid phase antibody-antigen-biotin-labeled antibody-avidin enzyme on the solid phase surface of the microwell plate. Add TMB colorimetric solution to produce a blue product, which eventually turns into yellow under the action of the stop solution. The absorbance (OD value) is measured at a wavelength of 450nm on an ELISA instrument. The absorbance (OD value) is positively correlated with the concentration of mouse phosphorylated AKT protein (p-AKT) in the sample to be tested. Fitting the calibrator curve can calculate the concentration of mouse phosphorylated AKT protein (p-AKT) in the sample.

This kit recognizes native mouse phosphorylated AKT protein (p-AKT) and has no cross-reactivity with structural analogs.

The coefficient of variation within and between plates was<10%.

The recovery rate is 85%-115%between.

1standard specification ELISA reader. 2, automatic plate washer. 3, oscillator. 4, series of adjustable pipettes and tips. When testing a large number of samples at one time, it is best to use a multi-channel pipette.

Recommended sample dilution plan: It is recommended that teachers conduct preliminary experiments to explore the optimal dilution multiple of the sample before conducting formal experiments.All reagents and components should be returned to room temperature first. For calibrators, quality control products, and samples, duplicate wells are recommended. 12. Prepare the working solutions of various components of the kit according to the method described in the previous instructions. 2, take out the required strips from the aluminum foil bag, seal the remaining strips in a ziplock bag and put them back in the refrigerator. 3, set up calibration wells, sample diluent wells, blank wells and sample wells, and add different concentrations of calibration wells to each calibration well50μL, add sample diluent to the sample diluent well50μL, no addition is made to the blank wells, and the sample wells are added with the sample to be tested50μLBiotin antibody was added to each well except the blank well.100uL, cover the reaction plate with a sealing film,37Incubate in a water bath or incubator away from light60min. 4, remove the sealing film, discard the liquid, pat dry on absorbent paper, fill each well with washing solution, and let it stand1min, shake off the detergent, pat dry on absorbent paper, and repeat this process.5If you use an automatic plate washer, please follow the plate washer operating procedures to wash the plate and add soaking30sAfter washing the plate, pat the plate dry on a clean, lint-free paper before adding the substrate. 5, except for the blank well, addHRPLabeled avidin100uL, cover the reaction plate with a sealing film,37Incubate in a water bath or incubator away from light20min. 6, repeat steps4. 7, add to all wellsTMBColor development solution100μL. Cover the reaction plate with a sealing film.37Incubate in a water bath or incubator away from light15min. 8, add stop solution to all wells50μL,exist450nmRead the absorbance of each well on a wavelength microplate reader (ODvalue).

1, This kit is for in vitro research use only and is not for clinical diagnosis. 2, Please wear lab coats and latex gloves for protection during the test. Especially when testing blood or other body fluid samples, please follow the national biological laboratory safety protection regulations. 3, incubate strictly according to the specified time and temperature to ensure accurate results. All reagents must reach room temperature before use 20-25℃. Refrigerate the reagent immediately after use. 4Improper plate washing may lead to inaccurate results. Make sure to aspirate as much liquid as possible before adding substrate. Do not allow the wells to dry out during incubation. 5, remove the residual liquid and fingerprints on the bottom of the board, otherwise it will affect ODvalue. 6, the substrate developing solution should be colorless or very light in color. 7, avoid cross contamination of reagents and specimens to avoid erroneous results. 8, Avoid direct exposure to strong light during storage and incubation. 9equilibrate to room temperature before opening the sealed bag to prevent water droplets from condensing on the cold strips. 10, Any reaction reagent cannot come into contact with bleaching solvent or the strong gas emitted by bleaching solvent. Any bleaching component will destroy the biological activity of the reaction reagent in the kit. 11, the ELISA reader used for testing needs to be installed with the ability to detect 450±10nmWavelength filters with optical density range of 0-3.5It is recommended to use in advance 15Minutes to preheat. 12, Do not mix or replace the reagents in this kit with reagents from other batches or other sources. 13, used in the test EPBoth the tube and the pipette tip are for single use and mixing is strictly prohibited. 14, Do not use expired reagents.

①Problem description: The positive and negative control results are unstablePossible causes and corresponding countermeasures: 1.Inaccurate aspiration or addition of liquid——Check the pipette and tips 2.Equilibration time is too short——Ensure adequate balancing time 3.Incomplete washing——Ensure the washing time, washing times and liquid volume per well ②Problem description: The color is very weak or colorlessPossible causes and corresponding countermeasures: 1.Incubation time is too short——Ensure adequate incubation time 2.Incorrect test temperature——Use the recommended test temperature 3.Insufficient reagent volume or missed addition/Incorrect dilution——Check the aspiration and addition process to ensure that all reagents are added in order and in sufficient quantities. 4.Enzyme marker inactivation or substrate failure——Mix the enzyme conjugate and substrate and check and judge by rapid color development ③Problem description: Low readingsPossible causes and corresponding countermeasures: 1.The microplate reader is not set up correctly——Check wavelength and filter settings on the microplate reader/Turn on the ELISA reader in advance to preheat ④Problem description: Large coefficient of variationPossible causes and corresponding countermeasures: 1.Incorrect addition of liquid——Check the filling status ⑤Problem description: High background valuePossible causes and corresponding countermeasures: 1.The working concentration of the detection antibody is too high——Use the recommended dilution factor 2.Incomplete washing of ELISA plate——Ensure that each step is cleaned completely; if using an automatic plate washer, check whether all outlets are blocked; whether the washing solution provided in the kit is used 3.The washing liquid is contaminated——Prepare new lotion ⑥Problem description: Low sensitivityPossible causes and corresponding countermeasures: 1.ELISAImproper storage of the test kit——Store relevant reagents according to the instructions 2.Not terminated before reading——ODStop solution should be added to each well before reading.

12. Due to the limitations of existing conditions and scientific and technological levels, it is not possible to conduct comprehensive identification and analysis of all raw materials. This product may have certain quality and technical risks. 2、This kit was removed during the development process/Some endogenous interfering factors in biological samples have been reduced, but not all possible influencing factors have been removed. 3The final experimental results are closely related to factors such as the effectiveness of the reagents, the experimenter's related operations, and the experimental environment at the time. Our company is only responsible for the kit itself and is not responsible for the sample consumption caused by the use of the kit. Please fully consider the possible usage of the sample before use and reserve sufficient samples. 4, In order to achieve good experimental results, please only use the reagents provided in our company's test kits, do not mix with products from other manufacturers, and strictly follow the instructions. 52. Abnormal results may occur due to incorrect reagent preparation and incorrect parameter settings of the ELISA reader during the operation. Please read the instructions carefully and adjust the instrument before the experiment. 6Even if the same person performs the operation, different results may be obtained in two independent experiments. In order to ensure the reproducibility of the results, it is necessary to control the operation of each step in the experimental process. 7the test kit will undergo strict quality inspection before shipment. However, due to factors such as transportation conditions, differences in experimental equipment, etc., the user's test results may be inconsistent with the factory data. 82. This kit has not been compared with similar kits from other manufacturers or products that detect the same target object using different methods, so inconsistent test results cannot be ruled out. 9, The kit is for research use only. If it is used for clinical diagnosis or any other purpose, our company will not be responsible for any problems arising therefrom and will not bear any legal liability.