Operation method of β-secretase activity ELISA kit in rat brain
Procedure 1. Dilution of standard product: This kit provides one original standard product. The user can perform dilution in a small test tube according to the following chart. 40pg / ml No. 5 standard 150μl original standard added 150μl standard dilution 20pg / ml No. 4 standard 150μl standard 5 added 150μl standard dilution 10pg / ml No. 3 standard 150μl No. 4 standard Add 150μl standard dilution 5pg / ml No. 2 standard 150μl No. 3 standard add 150μl standard dilution 2.5pg / ml No. 1 standard 150μl No. 2 standard add 150μl standard dilution 2. Add sample : Set up blank wells (the blank control wells do not add samples and enzyme reagents, the rest of the operation is the same), standard wells, sample wells to be tested Add 50μl of the standard on the enzyme-coated plate accurately, add 40μl of sample diluent to the sample well, 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 the 30-fold concentrated washing solution with distilled water 30-fold and reserve for use. 5. Washing: Carefully remove the sealing film, discard the liquid, spin dry, fill each well with the washing solution, and let it stand for 30 seconds before discarding , 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 (at this time, the blue will turn 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 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 the water bath during dilution, and the results will not be affected during washing. 3. The sample adder should be used in each step of sample addition, and its 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 liquid and various wastes should be treated as infectious agents. 9. The components of different batches of this reagent shall not be mixed. 10. If it is different from the English manual, the English manual shall prevail.
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