This kit is for research use only
Detection range: 7.8 pg / ml-500 pg / ml
Minimum detection limit: 1.95 pg / ml
Specificity: This kit can detect natural or recombinant mouse NANOG at the same time, and is related to other
There is no cross-reactivity of the protein.
Validity: 6 months
Intended application: ELISA method for quantitative determination of mouse serum, plasma, cell culture supernatant or other related
NANOG content in biological fluids.
Explanation
1. Store the kit: -20 ℃ (when not in use for a long time); 2-8 ℃ (when used frequently).
2. The concentrated washing liquid will have salt precipitation at low temperature, and it can be heated and dissolved in the water bath when diluted.
3. There may be inconsistencies between the Chinese and English instructions. Please refer to the English instructions.
4. There may be a little water-like substance in the well of the enzyme-linked plate just opened. This is a normal phenomenon and will not be wrong.
The test results have no effect.
Experimental principle
The microtiter plate is coated with purified antibody to make a solid-phase carrier.
Add samples or standards, biotinylated anti-NANOG antibody, HRP-labeled affinity to the wells in sequence
After thorough washing, it is developed with the substrate TMB. TMB is converted into peroxidase
Blue, and converted into the final yellow under the action of acid. The shade of color and the NANOG in the sample
Was positively correlated. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the sample concentration was calculated.
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Kit composition and reagent preparation
1. Assay plate: one piece (96 wells).
2. Standard (Standard): 2 bottles (lyophilized product).
3. Sample Diluent: 1 × 20ml / bottle.
4. Biotin-antibody Diluent: 1 × 10ml / bottle.
5. Horseradish peroxidase labeled avidin diluent (HRP-avidin Diluent) 1 × 10ml / bottle.
6. Biotin-antibody: 1 × 120μl / vial (1: 100).
7. Horseradish peroxidase-labeled avidin (HRP-avidin): 1 × 120 μl / vial (1: 100).
8. Substrate solution (TMB Substrate): 1 × 10ml / bottle.
9. Wash Buffer: 1 × 20ml / bottle, each bottle is diluted 25 times with distilled water.
10. Stop Solution (Stop Solution): 1 × 10ml / bottle (2N H2SO4).
Reagents and equipment needed but not provided
1. Standard Specification Microplate Reader
2. High-speed centrifuge
3. Electric heating thermostat incubator
4. Clean test tubes and Eppendof tubes
5. Series adjustable pipettes and tips. When testing more samples at one time, it is best to use multi-channel pipettes
6. Distilled water, volumetric flask, etc.
Collection and preservation of specimens
1. Serum: Whole blood samples should be left at room temperature for 2 hours or overnight at 4 ° C and centrifuged at 1000g for 20 minutes
Take the supernatant for testing, or store the specimen at -20 ℃ or -80 ℃, but avoid repeated freezing and thawing.
2. Plasma: EDTA or heparin can be used as anticoagulant, within 2-8 ° C 1000 within 30 minutes after specimen collection
g Centrifuge for 15 minutes, or store the specimen at -20 ℃ or -80 ℃, but avoid repeated freezing and thawing.
3. Cell culture supernatant or other biological specimens: centrifuge at 1000g for 20 minutes, take the supernatant for detection,
Or store the specimen at -20 ℃ or -80 ℃, but avoid repeated freezing and thawing.
Note: Hemolysis of specimens will affect the final test results, so hemolysis specimens should not be tested.
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Dilution principle of specimens:
First of all, we should know the approximate content of the sample to be tested through literature search, and determine the appropriate dilution factor.
Only when it is diluted to the range of the standard curve, the test result is accurate. During the dilution process,
Keep detailed records. When calculating the concentration at the end, it was diluted "N" times, and the concentration of the specimen should be multiplied by "N".
Standard dilution principle: 2 bottles, each bottle is diluted to 1ml with sample diluent before use, and capped
After standing for more than 10 minutes, then invert / rub repeatedly to help dissolve, the concentration is 500 pg / ml, do
After serial dilution, 500 pg / ml, 250 pg / ml, 125 pg / ml, 62.5 pg / ml,
31.2 pg / ml, 15.6 pg / ml, 7.8 pg / ml, the sample dilution is directly used as the standard concentration of 0 pg / ml,
Prepare within 15 minutes before use.
For the preparation of 250 pg / ml standards: take 0.5ml (not less than 0.5ml) of the above standards at 500 pg / ml
The product is added to an Eppendorf tube containing 0.5ml of sample diluent and mixed well, and the remaining concentrations can be deduced by analogy.
Dilution principle of biotinylated antibody:
Dilute with biotinylated antibody diluent immediately before use.
The total amount of preparation required (100μl per well), the actual preparation should be more 0.1-0.2ml. Such as 10μl biotin
Prepare the ratio of labeled antibody plus 990 μl of biotin-labeled antibody dilution, mix gently, before use
Prepare within one hour.
Dilution principle of horseradish peroxidase-labeled avidin:
Dilute with horseradish peroxidase-labeled avidin dilution before use, according to the pre-calculated before dilution
The total amount of preparation required for each experiment (100μl per well), the actual preparation should be more 0.1-0.2ml. Such as
10μl horseradish peroxidase labeled avidin diluted with 990μl horseradish peroxidase labeled avidin
Prepare the liquid ratio, mix gently, and prepare within one hour before use.
Steps
Before starting the experiment, please configure all reagents in advance. When the reagents or samples are diluted, they must be mixed well.
Try to avoid blistering. A standard curve should be made for each test. If the sample concentration is too high, use the sample
The dilution solution is diluted so that the sample conforms to the detection range of the kit.
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1. Add sample: set blank hole, standard hole and sample hole to be tested respectively. Add 100μl of sample diluent to blank wells,
Add 100μl of the standard product or the sample to be tested to the remaining hole, be careful not to have bubbles, add the sample to the sample
At the bottom of the well of the microplate, try not to touch the wall of the well, gently shake and mix well, add a cover or film to the microplate,
Incubate at 37 ° C for 120 minutes.
To ensure the validity of the experimental results, please use a new standard solution for each experiment.
2. Discard the liquid and spin dry without washing. Add 100μl of biotinylated antibody working solution to each well (take 1μl
Prepare a ratio of biotin-labeled antibody plus 99μl of biotin-labeled antibody dilution, mix gently,
Prepared within one hour before use), 37 ° C, 60 minutes.
3. After incubating for 60 minutes, discard the liquid in the well, spin dry, wash the plate 3 times, soak for 1-2 minutes each time,
200μl / well, spin dry.
4. Add horseradish peroxidase-labeled avidin working solution to each well (same as biotin-labeled antibody working solution)
100 μl, 37 ° C, 60 minutes.
5. After incubating for 60 minutes, discard the liquid in the hole, spin dry, wash the plate 5 times, soak for 1-2 minutes each time,
200μl / well, spin dry.
6. Add 90μl of substrate solution to each well in sequence, and develop color in the dark at 37 ° C (within 30 minutes, the standard is visible to the naked eye)
The first 3-4 wells of the quasi-products have a clear gradient blue, and the latter 3-4 wells are not obvious, then they can be terminated).
7. Add 50μl of stop solution to each well in sequence to stop the reaction (in this case, the blue color turns to yellow). Stop solution
The order of addition should be the same as the order of addition of substrate solution. In order to ensure the accuracy of the experimental results, the bottom
The stop solution should be added as soon as possible after the reaction time.
8. Measure the optical density (OD value) of each well in sequence using an enzyme-linked instrument at a wavelength of 450 nm. After adding stop solution
Test within 15 minutes.
Experimental remarks
1. When using the kit for the first time, the user should centrifuge various reagent tubes for a few minutes
Tube bottom.
2. Leave one well for each experiment as a blank zero-adjusting well. No reagents are added to this well, only the substrate is added at the end to dissolve
Liquid and 2N H2SO4. Use this hole to adjust the OD value to zero first during measurement.
3. To prevent the sample from evaporating, place the reaction plate in a closed box covered with a damp cloth during the test.
Cover or cover.
4. Store unused microplates or reagents at 2-8 ° C. Standards, biotinylated antibodies
Working solution, horseradish peroxidase-labeled avidin working solution, please configure and use according to the required amount. please
Do not reuse diluted standards, biotinylated antibody working solution, or horseradish peroxide
Enzyme-labeled avidin working solution.
5. It is recommended to set double-hole measurement when testing samples to ensure the accuracy of the test results.
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Washing method
Manual plate washing method: aspirate (do not touch the wall) or shake off the liquid in the enzyme plate; on the experimental table
Paved with several layers of absorbent paper, and slamming the microtiter plate down several times; put the recommended wash buffer at least 0.3ml
Fill the hole and soak for 1-2 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 skillfully.
Calculation
Taking the concentration of the standard as the abscissa (logarithmic coordinate), the OD value is the ordinate (common coordinate),
Draw a standard curve on the graph paper, and find out the corresponding concentration from the standard curve according to the OD value of the sample;
Multiply by the dilution factor; or use the standard concentration and OD value to calculate the linear regression of the standard curve
Formula, substitute the OD value of the sample into the equation, calculate the sample concentration, and multiply it by the dilution factor, that is
The actual concentration of the sample.
Precautions
1. These operating instructions apply to the 48T kit, but all reagents in the 48T kit are halved.
2. When mixing the protein solution, it should be as gentle as possible to avoid foaming.
3. The washing process is very important. Insufficient washing can easily cause false positives.
4. It is best to control the sampling time within 5 minutes. If there are many specimens, it is recommended to use a volley gun to add samples.
5. Please make a standard curve at the same time every measurement.
6. If the content of the substance to be tested in the specimen is too high, please dilute it before measuring, please multiply it by the dilution when calculating
multiple.
7. When preparing standard products and testing solution working fluids, please prepare with corresponding diluent, not to be confused.
8. Please keep the substrate away from light.
9. Do not replace the reagents in the kit with reagents from other manufacturers.
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