Plant Folic Acid (FA) Enzyme Linked Immunoassay (ELISA) Kit Instructions for Use This reagent is for research purposes only: This kit is used to determine the content of folate (FA) in plant tissues, cells and other related samples. Experimental principle: This kit uses the double antibody sandwich method to determine the level of plant folic acid (FA) in the specimen. The microplate was coated with purified plant folic acid (FA) antibody to make a solid phase antibody. Folic acid (FA) was added to the monoclonal antibody-coated microwells in turn, and then combined with HRP labeled folic acid (FA) antibody to form an antibody -Antigen-enzyme labeled antibody complex, after thorough washing, add substrate TMB to develop color. TMB is converted into blue under the catalysis of HRP enzyme, and into the final yellow under the action of acid. The color depth is positively correlated with the folic acid (FA) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of plant folic acid (FA) in the sample was calculated by a standard curve. Kit composition: Kit composition 48-well configuration 96-well configuration Preservation manual 1 copy 1 copy sealing film 2 pieces (48) 2 pieces (96) sealed bag 1 piece 1 enzyme-coated plate 1 × 48 1 × 96 2 Store standard at -8 ℃: 13.5μg / L 0.5ml × 1 bottle 0.5ml × 1 bottle at 2-8 ℃, store standard dilution 1.5ml × 1 bottle 1.5ml × 1 bottle at 2-8 ℃, store enzyme label reagent 3 ml × 1 bottle of 6 ml × 1 bottle of sample diluent 3 ml at 2-8 ° C × 1 bottle of 6 ml × 1 bottle of 2-8 ° C storage of developer A solution 3 ml × 1 bottle of 6 ml × 1 bottle of 2-8 ° C Storage Developer B solution 3 ml × 1 bottle 6 ml × 1 bottle at 2-8 ° C Storage stop solution 3ml × 1 bottle 6ml × 1 bottle 2-8 ° C Concentrated washing solution (20ml × 20 times) × 1 bottle (20ml × 30 times) × 1 bottle at 2-8 ° C to store specimens: 1. The specimens should be extracted as soon as possible after collection. The extraction should be carried out according to relevant literature. Experiments should be conducted as soon as possible after extraction. If the test cannot be performed immediately, the specimen can be stored at -20 ℃, but repeated freezing and thawing should be avoided. 2. The sample containing NaN3 cannot be detected because NaN3 inhibits the activity of horseradish peroxidase (HRP). Operation steps: 1. Dilution and loading of standard products: set 10 standard wells on the enzyme-coated plate, add 100 μl of standard products to the first and second wells, and then add them to the first and second wells Add 50μl of standard diluent and mix well; then take 100μl from the first and second wells respectively to the third and fourth wells, and then add 50μl of standard diluent to the third and fourth wells, Mix well; then take 50μl each in the third and fourth wells and discard it, then add 50μl each to the fifth and sixth wells, and then add 50ul of the standard dilution solution to the fifth and sixth wells, Mix; after mixing, take 50μl from the fifth and sixth wells and add them to the seventh and eighth wells respectively. Then add 50μl of the standard dilution solution to the seventh and eighth wells respectively. 7. Take 50μl from the eighth and eighth wells and add them to the ninth and tenth wells respectively. Then add 50μl of the standard dilution solution to the ninth and tenth wells respectively. After mixing, take 50μl from the ninth and tenth wells and discard. (After dilution, the volume of each well is 50μl, and the concentration is 9μg / L, 6μg / L, 3μg / L, 1.5μg / L, 0.75μg / L). 2. Adding samples: set up blank wells (the blank control wells do not add samples and enzyme reagents, the rest of the steps are the same) and the sample wells to be tested. Add 40μl of sample diluent to the test sample well on the enzyme-coated plate, and then add 10μl of the sample to be tested (the final dilution of the sample is 5 times). Add the sample and add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, shake gently to mix. 3. Incubation: Seal the plate with a sealing plate and incubate at 37 ° C for 30 minutes. 4. Mixing solution: Dilute 30 times (20 times of 48T) concentrated washing solution with distilled water 30 times (20 times of 48T) and then reserve. 5. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with the washing liquid, let it stand for 30 seconds and then discard, repeat this 5 times, pat dry. 6. Add enzyme: add 50μl of enzyme label reagent to each well, except blank well. 7. Incubation: The operation is the same as 3. 8. Washing: The operation is the same as 5. 9. Color development: add 50μl of developer A to each well, then add 50μl of developer B, mix gently, and develop color at 37 ° C in the dark 15 minutes. 10. Termination: Add 50 μl of stop solution to each well to stop the reaction (in this case, the blue color turns to yellow). 11. Determination: Measure the absorbance (OD value) of each well in sequence with the blank air conditioner at zero and 450 nm wavelength. The measurement should be carried out within 15 minutes after adding the stop solution. Note: 1. The kit should be taken out of the refrigerated environment and should be equilibrated at room temperature for 15-30 minutes before use. If the enzyme-coated plate is not used up after opening, the strip should be stored in a sealed bag. 2. Crystals may be precipitated in the concentrated washing liquid, which can be heated and dissolved in the water bath during dilution, and the results will not be affected during washing. 3. The sampler should be used at each step of sample addition, and the accuracy should be regularly checked to avoid test errors. 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. If the content of the substance to be tested in the specimen is too high (the OD value of the sample is greater than the OD value of the first well of the standard well), please dilute it with a certain multiple of the sample diluent (n times) before measuring, and finally multiply by the total dilution Multiple (× n × 5). 5. The sealing film is limited to one-time use to avoid cross contamination. 6. Please keep the substrate away from light. 7. Strictly follow the instructions, and the test results must be determined based on the reading of the microplate reader. 8. All samples, washing solutions and various wastes should be treated as infectious agents. 9. The components of different batches of this reagent shall not be mixed. 10. If there is any difference with the English manual, the English manual shall prevail. Calculation: Taking the concentration of the standard as the abscissa and the OD as the ordinate, 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 The linear regression equation of the standard curve is calculated by the concentration and OD value of the sample, the OD value of the sample is substituted into the equation, the sample concentration is calculated, and then multiplied by the dilution factor, which is the actual concentration of the sample. (This picture is for reference only) Kit performance: 1. The linear regression coefficient of the sample and the expected concentration correlation coefficient R value is above 0.95. 2. The batch and batch see should be less than 9% and 11% respectively. Detection range: 0.5μg / L -10μg / L Storage conditions and expiration date: 1. Kit storage :; 2. Validity period: 6 months Shanghai Yuping Biological Technology Co., Ltd. mainly deals with ELISA kits of various brands and grades, with quality assurance and perfect after-sales service. And provide free generation testing. Serving universities and immunology research units. Technicians serve you better.
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