The detection range of fish myostatin (MSTN) kit (ELISA) is simple, accurate, and easy to operate. It is trusted and supported by many researchers. l This kit is used for quantitative determination of fish myostatin (MSTN) in body fluids, cavity fluids, tissue homogenates and related liquid samples in vitro.
l Validity: 6 months
l Storage conditions: 2-8 ° C
l This kit is for scientific research purposes only and should not be used for clinical diagnosis.
Experimental principle The kit uses a double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). The coated microcapsules of the pre-coated fish myostatin (MSTN) capture antibody were sequentially added to the specimen, the standard, and the HRP-labeled detection antibody, and incubated and thoroughly washed. Using the substrate TMB to develop color, TMB is converted to blue under the catalysis of peroxidase and converted to the final yellow color by the action of an acid. The color depth is positively correlated with fish myostatin (MSTN) in the sample. The absorbance (OD value) was measured at 450 nm using a microplate reader to calculate the sample concentration.
Sample processing and requirements
1. Serum: Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or 4 ° C overnight, then centrifuge at 1000 × g for 20 minutes, take the supernatant, or place the supernatant at -20 ° C or -80 Store at °C, but avoid freezing and thawing repeatedly.
2. Plasma: Collect specimens with EDTA or heparin as anticoagulant, and centrifuge the specimens at 1000×g for 15 minutes at 2-8 °C within 30 minutes after collection. The supernatant can be detected or the supernatant can be placed. Store at -20 ° C or -80 ° C, but avoid repeated freezing and thawing.
3. Tissue homogenization: Wash the tissue with pre-cooled PBS (0.01M, pH=7.4) to remove residual blood (the red blood cells in the homogenate will affect the measurement results), and weigh the tissue after weighing. The shredded tissue and the corresponding volume of PBS (generally in a weight ratio of 1:9, such as 1g of tissue sample corresponding to 9mL of PBS, the specific volume can be adjusted according to the needs of the experiment, and record. Recommended to add in PBS The protease inhibitor) was added to a glass homogenizer and thoroughly ground on ice. To further lyse tissue cells, the homogenate can be sonicated or repeatedly frozen and thawed. Finally, the homogenate was centrifuged at 5000×g for 5-10 minutes, and the supernatant was taken for detection.
4. Cell culture supernatant or other biological specimens: centrifuge at 1000 × g for 20 minutes, take the supernatant to detect, or store the supernatant at -20 ° C or -80 ° C, but avoid repeated freezing and thawing.
Note: Specimen hemolysis will affect the final test results, so hemolysis specimens should not be tested.
Reagents and equipment that are not provided
- Microplate reader (450nm)
- High-precision sampler and tip: 0.5-10uL, 2-20uL, 20-200uL, 200-1000uL
- 37 ° C incubator
- Distilled or deionized water
Kit composition
| name | 96-well configuration | 48-hole configuration | Remarks |
| Microporous plate | 12 holes × 8 | 12 holes × 4 | no |
| Standard | 0.3mL*6 tube | 0.3mL*6 tube | no |
| Sample diluent | 6mL | 3mL | no |
| Detection antibody-HRP | 10mL | 5mL | no |
| 20× washing buffer | 25mL | 15mL | Dilute according to the instructions |
| Substrate A | 6mL | 3mL | no |
| Substrate B | 6mL | 3mL | no |
| Stop solution | 6mL | 3mL | no |
| Sealing film | 2 sheets | 2 sheets | no |
| Instruction manual | 1 serving | 1 serving | no |
| Ziplock bag | 1 | 1 | no |
Remarks:
1. The concentration of the standard is: 50, 25, 12.5, 6.25, 3.125, 1.5625 ng/mL
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, and 50 μL of the sample can be directly loaded during the experiment. When part of the sample value exceeds the maximum standard concentration, the sample may be diluted with the sample dilution before performing the experiment.
Precautions
- Incubation is carried out strictly in accordance with the specified time and temperature to ensure accurate results. All reagents must reach room temperature 20-25 °C before use. Store the reagents refrigerated immediately after use.
- Incorrect cleaning can result in inaccurate results. Make sure to drain the liquid in the well as much as possible before adding the substrate. Do not let the micropores dry during the incubation.
- Eliminate residual liquid and fingerprints at the bottom of the board, otherwise it will affect the OD value.
- The substrate coloring solution should be colorless or very light, and the substrate liquid that has turned blue cannot be used.
- Avoid cross-contamination of reagents and specimens to avoid erroneous results.
- Avoid direct exposure to strong light during storage and incubation.
- After balancing to room temperature, the sealed bag was opened to allow water-repellent drops to coagulate on the cold strip.
- Any reagents should not be exposed to strong gases emitted by bleaching solvents or bleaching solvents. Any bleaching component will destroy the biological activity of the reagents in the kit.
- Expired products cannot be used.
- If the disease is likely to spread, all samples should be managed and the sample and test device processed in accordance with the prescribed procedures.
The reagent preparation kit should be taken out of the refrigerated environment and should be equilibrated at room temperature before use.
Dilution of 20× Wash Buffer: Distilled water was diluted 1:20, ie 1 part of 20× wash buffer plus 19 parts of distilled water.
Steps
- The required slats were taken out from the aluminum foil pouch after equilibrating for 20 min at room temperature, and the remaining slats were sealed back to 4 ° C with a ziplock bag.
- Set standard and sample wells, standard wells with different concentrations of standard 50μL;
- Add 50 μL of the sample to be tested to the sample well; blank holes are not added.
- In addition to the blank wells, 100 μL of horseradish peroxidase (HRP)-labeled detection antibody was added to each well of the standard well and the sample well, and the reaction well was sealed with a sealing plate, and incubated at 37 ° C in a water bath or incubator for 60 min.
- Discard the liquid, pat dry on the absorbent paper, fill each well with the washing solution (350 μL), let stand for 1 min, remove the washing solution, pat dry on the absorbent paper, and repeat the washing 5 times (can also be washed with a washing machine) .
- 50 μL of each of the substrates A and B was added to each well, and incubated at 37 ° C for 15 min in the dark.
- 50 μL of the stop solution was added to each well, and the OD value of each well was measured at a wavelength of 450 nm within 15 min.
The experimental results are calculated by taking the OD value of the measured standard as the abscissa and the concentration of the standard as the ordinate. The standard curve is drawn on the coordinate paper or with the relevant software, and the linear regression equation is obtained, and the OD value of the sample is substituted into the equation. Calculate the concentration of the sample.
Kit performance
- Detection range: 1.5625 ng/mL – 50 ng/mL.
- Sensitivity: The minimum detection concentration is less than 0.1 ng/mL.
- Specificity: Does not cross-react with other soluble structural analogs.
- Repeatability: The intra-plate variation coefficient is less than 10%, and the inter-plate variation coefficient is less than 15%.
It is indicated that due to the existing conditions and the level of science and technology, it is not possible to comprehensively identify and analyze all the raw materials provided by all suppliers. This product may have certain quality and technical risks.
- The final experimental results are closely related to the effectiveness of the reagents, the experimenter's related operations, and the experimental environment at the time. Be sure to prepare sufficient specimen backups.
- Different batches of the same product may have a slight difference, such as: detection limit, sensitivity and color development time, etc., please carry out the experimental operation according to the instructions in the kit. The electronic version of the website is for reference only.
- Only the reagents used in this kit can be used to ensure the test results. Do not mix products from other manufacturers. The best test results will only be obtained if the experimental instructions of this kit are strictly followed.
- The 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.
- Tissue homogenates or cell extracts prepared using chemical lysates may cause bias in ELISA results due to the introduction of certain chemicals.
- If the sample is a cell culture supernatant, there are many factors that interfere with such samples, such as cell status, cell number, sampling time, etc., so there may be cases where detection is not possible.
- Certain natural or recombinant proteins, including prokaryotic and eukaryotic recombinant proteins, may not be detected because they do not match the detection and capture antibodies used in this product.
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