This kit is for research use only
Detection range: 56.1 pg / ml-3600 pg / ml
Minimum detection limit: 14 pg / ml
Specificity: This kit can detect natural or recombinant human DSIP at the same time, and is not related to other related proteins
Cross reaction.
Validity: 6 months
Intended application: ELISA method for quantitative determination of human serum, plasma, cell culture supernatant or other related
The content of DSIP in the liquid.
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
Coat the microtiter plate with purified antibody to make a solid phase carrier, and then to the microwells coated with anti-DSIP antibody
Add specimens or standards, biotinylated anti-DSIP antibody, HRP-labeled avidin, after thorough
After the bottom wash, the color is developed with the substrate TMB. TMB is converted into blue under the catalysis of peroxidase, and in
Under the action of acid, it turns into the final yellow. The color depth is positively correlated with the DSIP in the sample. With enzymes
The standard instrument measures the absorbance (OD value) at a wavelength of 450 nm and calculates the sample concentration.
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.
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5. 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 1000 xg for 20 minutes
Clock, take the supernatant for testing, or store the specimen at -20 ℃ or -80 ℃, but should avoid repeated freezing
melt.
2. Plasma: EDTA or heparin can be used as anticoagulant, within 2-8 ° C within 30 minutes after specimen collection
Centrifuge at 1000 xg for 15 minutes, or store the specimen at -20 ℃ or -80 ℃, but avoid repeated freezing
melt.
3. Cell culture supernatant or other biological specimens: centrifuge at 1000 xg for 20 minutes, take the supernatant for inspection
Measure 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.
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 with sample diluent to 1ml before use, after capping
Let stand for more than 10 minutes, then invert / rub repeatedly to help dissolve. Its concentration is 3600 pg / ml.
After the column ratio is diluted, they are respectively diluted 3600 pg / ml, 1800 pg / ml, 900 pg / ml, 450 pg / ml, 225
pg / ml, 112.2 pg / ml, 56.1 pg / ml, the sample dilution is directly used as the standard concentration of 0 pg / ml, ready for use
Prepare within the first 15 minutes.
If preparing 1800 pg / ml standard: take 0.5ml (not less than 0.5ml) 3600 pg / ml of the above standard
The quasi-product is added to the Eppendorf tube containing 0.5ml of sample diluent and mixed well.
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Push.
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.
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,
350μl / per 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,
350μl / per 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 have no obvious gradient and 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
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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.
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. When mixing protein solutions, try to be as gentle as possible to avoid foaming.
2. The washing process is very important. Insufficient washing can easily cause false positives.
3. 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.
4. Please make the standard curve at the same time of each measurement, it is better to make the complex hole.
5. If the content of the substance to be tested in the specimen is too high, please first dilute it and then determine it.
multiple.
6. When preparing standard products and testing solution working fluid, please prepare with corresponding diluent, not to be confused.
7. Please keep the substrate away from light.
8. Do not replace the reagents in the kit with reagents from other manufacturers.
Three to four hours after the completion of the tattoo, clean the tattoo with warm water, use a bath, then dry with a sanitary towel, keep dry, apply the care cream, after cleaning every day, apply once.
Tattoo attention points - maintenance
1. After completing the tattoo, clean the whole part with color toner, apply color anti-inflammatory gel, then wrap the bandage and keep it for at least two hours;
2. Undo the bandage, clean the tattoo with color toner and blot the water dry;
3, apply color anti-inflammatory gel, and wipe off the excess, color anti-inflammatory gel four times a day;
4, keep the tattoo part moist and breathable, no need to bandage;
5. Swimming or long time immersion in water is prohibited within one week;
6. Avoid excessive exposure to sunlight for one month;
7, can not scratch or tear off the scab part;
8, can be used to pat or wipe alcohol method to reduce itching;
9. Wear loose clothing.
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