Rat Melatonin (MT) ELISA Kit

Rat Melatonin (MT) ELISA Kit

lThis kit is used for in vitro quantitative detectionSerum, plasma, tissue homogenate, and related liquid samplesmiddleRat melatonin（MT）The content.

lValidity period: 6 months

lStorage conditions:2-8℃

lThis kit is for scientific research purposes only and should not be used for clinical diagnosis

Experimental Principle

The reagent kit adopts a double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). Add the sample, standard, and HRP labeled detection antibody to the pre coated micro well of rat melatonin (MT) capture antibody, incubate and wash in sequence. Using substrate TMB for color development, TMB is converted to blue under the catalysis of peroxidase and to the final yellow under the action of acid. The depth of color is positively correlated with rat melatonin (MT) in the sample. Measure the absorbance (OD value) at a wavelength of 450nm using an enzyme-linked immunosorbent assay (ELISA) reader and calculate the sample concentration.

Sample processing and requirements

1. serum：Place the whole blood samples collected in the serum separation tube at room temperature for 2 hours or overnight at 4 ℃, then centrifuge at 1000 × g for 20 minutes to obtain the supernatant, or store the supernatant at -20 ℃ or -80 ℃, but avoid repeated freezing and thawing.
2. plasma：Collect specimens using EDTA or heparin as anticoagulants, and centrifuge the specimens at 2-8 ℃ 1000 × g for 15 minutes within 30 minutes after collection. The supernatant can be collected for detection, or stored at -20 ℃ or -80 ℃, but repeated freezing and thawing should be avoided.
3. Tissue homogenate:Wash the tissue with pre cooled PBS (0.01M, pH=7.4) to remove residual blood (lysed red blood cells in the homogenate can affect the measurement results), weigh and cut the tissue into pieces. Mix the shredded tissue with the corresponding volume of PBS (usually in a weight to volume ratio of 1:9, for example, 1g of tissue sample corresponds to 9mL of PBS, the specific volume can be adjusted appropriately according to experimental needs, and records should be kept. Recommend adding protease inhibitors to PBS and grinding thoroughly on ice in a glass homogenizer. To further lyse tissue cells, the homogenate can be sonicated or subjected to repeated freeze-thaw cycles. Afterwards, centrifuge the homogenate at 5000 × g for 5-10 minutes, and take the supernatant for detection.

4. Cell culture supernatant or other biological specimens:Please centrifuge at 1000 × g for 20 minutes, take the supernatant for detection, or store the supernatant at -20 ℃ or -80 ℃, but avoid repeated freezing and thawing.

Note: Hemolysis of specimens can affect the results of subsequent tests, therefore hemolytic specimens are not suitable for this test.

Reagents and equipment that are needed but not provided

• ELISA reader (450nm)

• High precision sampler and nozzle: 0.5-10uL, 2-20uL, 20-200uL, 200-1000uL

• 37 ℃ constant temperature box

• Distilled water or deionized water

Kit components

Rat Melatonin (MT) ELISA Kitpreparenote：

1. The concentrations of the standard samples are: 80, 40, 20, 10, 5, and 2.5 pg/mL, respectively

2. After a large number of normal specimen tests, the normal concentration values of the specimens are within the detection range provided by the kit. During the experiment, 50 μ L of the sample can be directly taken and loaded. When some sample values exceed the maximum standard concentration, the sample dilution solution can be used to appropriately dilute the specimen before conducting the experiment.

Precautions

• Strictly follow the prescribed time and temperature for incubation to ensure accurate results. All reagents must reach room temperature of 20-25 ℃ before use. Immediately refrigerate and store the reagents after use.

• Incorrect board washing can lead to inaccurate results. Ensure to absorb as much liquid as possible from the well before adding the substrate. Do not let the micropores dry out during the incubation process.

• Eliminate residual liquid and fingerprints at the bottom of the board, otherwise it will affect the OD value.

• The substrate color solution should be colorless or very light in color, and the substrate solution that has turned blue cannot be used.

• Avoid cross contamination between reagents and specimens to prevent erroneous results.

• Avoid direct exposure to strong light during storage and incubation.

• After balancing to room temperature, open the sealing bag to prevent water droplets from condensing on the cold Flat noodles.

• Any reaction reagent should not come into contact with bleach solvents or the strong gases emitted by bleach solvents. Any bleaching component will destroy the biological activity of the reactants in the reagent kit.

• Expired products cannot be used.

• If there is a possibility of spreading the disease, all samples should be managed and processed according to the prescribed procedures and testing devices.

Reagent preparation

The reagent kit should be taken out of the refrigerated environment and equilibrated at room temperature before use.

Dilution of 20 x washing buffer: Dilute distilled water at a ratio of 1:20, that is, 1 part of 20 x washing buffer is added to 19 parts of distilled water.

operating steps

1. Take out the required Flat noodles from the aluminum foil bag after 20 min of room temperature balance, and seal the remaining Flat noodles with a self sealing bag and put it back at 4 ℃.

2. Set up standard wells and sample wells, and add 50 μ L of standard samples of different concentrations to each standard well;

3. Add 50 μ L of the test sample to the sample well; Blank holes are not added.

4. Except for blank wells, 100 μ L of horseradish peroxidase (HRP) labeled detection antibody was added to each well of the standard and sample wells. The reaction wells were sealed with a plate membrane and incubated at 37 ℃ in a water bath or constant temperature incubator for 60 minutes.

5. Discard the liquid, pat dry on absorbent paper, fill each well with washing solution (350 μ L), let it stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, repeat washing the board 5 times (or use a washing machine to wash the board).

6. Add 50 μ L of substrate A and B to each well, and incubate at 37 ℃ in the dark for 15 minutes.

7. Add 50 μ L of termination solution to each well and measure the OD value of each well at a wavelength of 450nm within 15 minutes.

Calculation of experimental results

Using the OD value of the measured standard as the horizontal axis and the concentration value of the standard as the vertical axis, draw a standard curve on a coordinate paper or with relevant software, and obtain a linear regression equation. Substitute the OD value of the sample into the equation to calculate the concentration of the sample.

Test kit performance

• Detection range: 2.5 pg/mL -80 pg/mL.

• Sensitivity: Low detection concentration less than 0.1 pg/mL.

• Specificity: Does not cross react with other soluble structural analogues.

• Repeatability: The intra plate coefficient of variation is less than 10%, and the inter plate coefficient of variation is less than 15%.

explanation

Due to the current conditions and scientific and technological level, it is not yet possible to comprehensively identify and analyze all raw materials provided by all suppliers, and there may be certain quality and technical risks associated with this product.

• The final experimental results are closely related to the effectiveness of the reagents, the relevant operations of the experimenter, and the experimental environment at that time. Please be sure to prepare sufficient sample backups.

• There may be slight differences in the detection limit, sensitivity, and color development time of the same product from different batches. Please refer to the instructions in the reagent kit for experimental operations. The electronic version of the instructions on the website is for reference only.

• Only by using all the reagents in this kit can the detection effect be guaranteed, and products from other vendors cannot be mixed. Only by strictly following the experimental instructions of this kit can we obtain * test results.

• Our company is only responsible for the reagent kit itself and is not responsible for the sample consumption caused by the use of the kit. Users are advised to fully consider the possible usage of the sample and reserve sufficient samples before use.

• The preparation of tissue homogenates or cell extracts using chemical lysates may result in bias in ELISA experimental results due to the introduction of certain chemicals.

• If the sample is a cell culture supernatant, there may be many interfering factors such as cell status, cell quantity, sampling time, etc., so there may be situations where it cannot be detected.

• Some natural or recombinant proteins, including prokaryotic and eukaryotic recombinant proteins, may not be detected due to mismatches with the detection and capture antibodies used in this product.