Human visfatin / visfatin (visfatin) ELISA method operation steps 1. Dilution of standard products: This kit provides one original standard, the user can perform dilution in a small test tube according to the following chart. 400pg / ml No. 5 standard 150Î¼l original standard added 150Î¼l standard dilution 200pg / ml No. 4 standard 150Î¼l standard 5 added 150Î¼l standard dilution 100pg / ml No. 3 standard 150Î¼l No. 4 standard Add 150Î¼l standard dilution 50pg / ml No. 2 standard 150Î¼l No. 3 standard add 150Î¼l standard dilution 25pg / ml No. 1 standard 150Î¼l No. 2 standard add 150Î¼l standard dilution 2. Add sample: Separately set blank wells (the blank control wells do not add samples and enzyme label reagents, the rest of the operation is the same), standard wells, sample wells to be tested. Accurately add 50Î¼l of the standard on the enzyme-coated plate, add 40Î¼l of sample diluent to the sample well, and then add 10Î¼l 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 Â° C for 30 minutes. 4. Mixing solution: dilute 20-fold concentrated washing solution with distilled water 20-fold and reserve for use 5. Washing: carefully peel off the sealing film, discard the liquid, spin dry, fill each well with the washing solution, and let it sit for 30 seconds , Repeat this 5 times, pat dry. 6. Add enzyme: add 50Î¼l 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 50Î¼l of developer A to each well, then add 50Î¼l of developer B, mix gently, and develop color at 37 Â° C in the dark 15 minutes. 10. Stop: Add 50Î¼l of stop solution to each well to stop the reaction (the blue color turns to yellow at this time). 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. Matters needing attention 1. The kit should be equilibrated at room temperature for 15-30 minutes before being taken out of the refrigerated environment. If the enzyme label coated plate is unopened, 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 a 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 a standard curve at the same time of each measurement, it is best to make a double hole. If the content of the test substance 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 first dilute it with a certain multiple (n times) of the sample diluent and then determine it. When calculating, please multiply the total dilution Multiple (Ã— n Ã— 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 liquids and various wastes should be treated as infectious agents. 9. The components of different batches of this reagent shall not be mixed.
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