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Analysis Manual of sheep follicle stimulating hormone (FSH) ELISA Kit

Your location: home page ?>? Technical literature Analysis Manual of sheep follicle stimulating hormone (FSH) ELISA kit

Brief introduction

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l? The kit is used for quantitative detection of serum, plasma, tissue, cell supernatant and related liquid samples in vitro Sheep follicle stimulating hormone FSH )The content of.
l? Validity: 6 months
l? Storage conditions: two -8℃
l? This kit is only used for in vitro study, not for clinical diagnosis
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Experimental principle
One step sandwich ELISA was used in the kit. The antibody to FSH was added to the follicle of the sheep, which was labeled with HRP. TMB was used as the substrate for color development. TMB was converted into blue under the catalysis of peroxidase and finally yellow under the action of acid. There was a positive correlation between the color and FSH in the samples. The absorbance (OD value) was measured at 450 nm wavelength by enzyme labeled instrument, and the concentration of the sample was calculated.
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Sample processing and requirements
1. ? serum : the whole blood sample should be placed at room temperature for 2 hours or 4 ℃ overnight, and centrifuged at 1000g for 20 minutes. The supernatant can be detected or stored at - 20 ℃ or - 80 ℃, but repeated freezing and thawing should be avoided.
2. ? plasma Methods: EDTA or heparin could be used as anticoagulant. The samples were centrifuged at 2-8 ° C 1000g for 20 minutes within 30 minutes after collection, or stored at - 20 ℃ or - 80 ℃, but repeated freezing and thawing should be avoided.
3. ? Homogenate tissue Methods: the tissues were washed with pre cooled PBS (0.01M, pH = 7.4) to remove the residual blood (the lysed red blood cells in the homogenate would affect the measurement results), and the tissues were cut into pieces after weighing. Cut the tissue and the corresponding volume of PBS (generally according to the weight volume ratio of 1:9, for example, 1g tissue sample corresponds to 9ml PBS, the specific volume can be adjusted according to the needs of the experiment, and the record should be made. It is recommended to add protease inhibitor into PBS) into glass homogenizer and grind it on ice. In order to further lyse the tissue cells, the homogenate can be broken by ultrasound or freeze-thaw repeatedly. Finally, the homogenate was centrifuged at 5000 × g for 5-10 minutes, and the supernatant was detected.
4. ? Cell culture supernatant or other biological specimens : 1000 g centrifugation for 20 minutes, the supernatant can be detected, or the specimen can be stored at - 20 ℃ or - 80 ℃, but repeated freezing and thawing should be avoided.
Note: hemolysis of the sample will affect the final test results, so it is not suitable to carry out this test on hemolytic samples.

Specimen treatment
1. The company is only responsible for the kit itself, not for the sample consumption caused by the use of the kit. Please fully consider the possible use of samples and reserve sufficient samples before use.
2. The sample content should be predicted before the experiment. If the sample concentration is too high, the sample should be diluted to make the diluted sample meet the detection range of the kit, and then multiply the corresponding dilution multiple. The samples were diluted with 0.01 mol / L PBS (pH = 7.0-7.2).
3. If the tested samples are not included in the samples listed in the instruction manual, it is recommended to conduct pre experiment to verify its effectiveness, and pay attention to the retention of samples.
4. Tissue homogenate or cell extract prepared by chemical lysate may lead to the deviation of ELISA results due to the introduction of some chemicals.
5. If the sample is cell culture supernatant, it may not be detected because of many interference factors, such as cell state, cell number, sampling time, etc.
6. Some natural proteins 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.
7. It is suggested that fresh samples should be used. If the storage time is too long, protein degradation or denaturation may lead to deviation of experimental results.
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Reagents and equipment required but not provided
1. ELISA (450nm)
2. High precision sampler and gun head: 0.5-10ul, 2-20ul, 20-200ul, 200-1000ul
3. 37 ℃ incubator
4. Distilled water or deionized water
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Kit composition
name
96 hole configuration
48 hole configuration
remarks
Microplate
8 holes × 12 pieces
8 holes × 6 pieces
nothing
Standard
0.3ml * 6 tubes
0.3ml * 6 tubes
nothing
Sample diluent
6mL
3mL
nothing
Detection of antibody HRP
10mL
5mL
nothing
20 × washing buffer
25mL
15mL
Dilute according to the instructions
Substrate a
6mL
3mL
nothing
Substrate B
6mL
3mL
nothing
Termination fluid
6mL
3mL
nothing
Sealing plate
2 sheets
2 sheets
nothing
instructions
1 copy
1 copy
nothing
Self sealing bag
1
1
nothing
prepare notes
1. The concentration of standard substance was 8, 4, 2, 1, 0.5, 0 mIU / ml
2. After a large number of normal samples were tested, the normal concentration values of the samples were within the detection range provided by the kit. During the experiment, 50 μ l sample can be directly taken for sample loading. When some sample values exceed the maximum standard concentration, the sample diluent can be used for appropriate dilution before the experiment.
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matters needing attention
1. Carry out temperature cultivation in strict accordance with the specified time and temperature to ensure accurate results. All reagents must reach room temperature 20-25 ℃ before use. Refrigerate the reagent immediately after use.
2. Incorrect plate washing can lead to inaccurate results. Before adding the substrate, make sure that the liquid in the hole is dried as much as possible. Do not let the micropores dry out in the process of warm cultivation.
3. Eliminate the residual liquid and finger print at the bottom of the plate, otherwise the OD value will be affected.
4. The substrate solution should be colorless or light color, and the substrate solution that has turned blue cannot be used.
5. Avoid cross contamination of test agent and sample to avoid wrong results.
6. Avoid direct exposure to strong light during storage and incubation.
7. After balancing to room temperature, open the sealing bag to prevent water droplets from condensing on the cold strip.
8. Any reaction reagent should not contact with bleaching solvent or strong gas emitted by bleaching solvent. Any bleaching component will destroy the biological activity of the reagent in the kit.
9. Do not use expired products.
10.? If there is a risk of disease transmission, all samples should be managed and treated according to the specified procedures
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Reagent preparation
The kit should be taken out from the refrigerated environment and should be balanced at room temperature before use.
Dilution of 20 × washing buffer: dilute distilled water by 1:20, that is, 1 part of 20 × washing buffer solution plus 19 parts of distilled water.
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Operation steps
1. Take out the required strips from the aluminum foil bags after 20 minutes of room temperature balance, and put the remaining strips back to 4 ℃ with self sealing bags.
2. Set up the standard sample hole and sample hole, and add 50 μ l standard substance of different concentration into the standard sample hole;
3. Add 50 μ l sample into the sample hole; do not add the blank hole.
4. In addition to the blank hole, 100 μ l HRP labeled antibody was added into each well of standard sample hole and sample hole. The reaction hole was sealed with sealing plate membrane, and incubated in 37 ℃ water bath or incubator for 60 min.
5. Discard the liquid, pat dry on the absorbent paper, fill each hole with washing solution (350 μ L), let stand for 1 min, throw away the washing liquid, pat dry on the absorbent paper, and wash the plate for 5 times in this way (also use washing machine to wash the plate).
6. 50 μ l substrate a and 50 μ L B were added into each well and incubated for 15 min at 37 ℃.
7. Add 50 μ l termination solution into each well, and within 15 minutes, determine the OD value of each pore at 450 nm.
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Calculation of experimental results
Taking the OD value of the standard substance as the abscissa and the concentration value of the standard as the ordinate, the standard curve is drawn on the coordinate paper or by using relevant software, and the linear regression equation is obtained. The OD value of the sample is substituted into the equation to calculate the concentration of the sample.
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Performance of kit
1. Detection range: 0.25 mIU / ml – 8 mIU / ml.
2. Sensitivity: the minimum detection concentration is less than 0.1 mIU / ml.
Specificity: no cross reaction with other soluble analogues.
4. Repeatability: the coefficient of variation within the plate is less than 10%, and the coefficient of variation between plates is less than 15%.
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explain
1. Due to the existing conditions and the level of science and technology can not fully identify and analyze all raw materials provided by all suppliers, this product may have certain quality and technical risks.
2. The final experimental results are closely related to the effectiveness of the reagent, the relevant operation of the experimenter and the experimental environment at that time. Please be sure to prepare sufficient specimen backup.
3. Different batches of the same product may have some differences, such as detection limit, sensitivity and color development time. Please carry out the experiment according to the instructions in the kit. The electronic version of the manual on the website is only for reference.
4. Only when all the matching reagents of this kit are used can the detection effect be guaranteed, and the products of other manufacturers cannot be mixed. The best detection results can be obtained only by strictly following the experimental instructions of this kit.
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Question and answer
If the experimental effect is not good, please take photos of the color development results in time, keep the strips and unused reagents, and keep them properly, and then contact our technical support to solve the problem for you. You can also refer to the following materials:
problem
Possible causes
Solution
Standard curve deviation
Inadequate absorption and washing
Adequate absorption and washing
Inaccurate transfer
Check and correct the pipette
Low precision
Inadequate washing
Wash and soak thoroughly according to instructions
Insufficient mixing and insufficient reagent absorption
Mix well and absorb the reagent
Container and reusable film
When using the sampler, replace the syringe with a new suction head, use a new container and cover the film
Inaccurate sample addition
Check and correct the pipette
O. Low value D
The amount of reagent added per well is not accurate
Correct the pipette and add reagent accurately
Incorrect warming time
Sufficient time for warm breeding
The temperature of incubation is not correct
The reagent should be balanced to room temperature and accurate incubation temperature should be ensured
Enzyme marker or substrate failure
By mixing the enzyme marker and substrate, the color should appear quickly to check
No termination solution was added
Add the termination solution according to the instruction
Over reading time reading
Read within the reading time recommended in the manual
Sample value
Incorrect sample storage
Store samples correctly and use fresh samples for experiments
Incorrect sample collection and handling methods
Adopt the correct method of sample collection and treatment
The content of the substance to be tested is low in the sample
Using fresh samples, repeat the experiment
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