Operation steps of rat inducible nitric oxide synthase:
1. Dilution of standard products: This kit provides one original standard product. The user can perform dilution in small test tubes according to the following chart.
12 IU / L
Standard No. 5
150μl of original standard is added to 150μl standard dilution
6 IU / L
Standard 4
150μl Standard No. 5 is added to 150μl standard dilution
3 IU / L
Standard 3
150μl Standard No. 4 is added to 150μl Standard Diluent
1.5 IU / L
Standard No. 2
150μl Standard No. 3 is added to 150μl Standard Diluent
0.75 IU / L
Standard 1
150μl Standard No. 2 is added to 150μl Standard Diluent
2. Sample addition: 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. Accurately add 50μl of the standard on the enzyme-coated plate, add 40μl of sample diluent to the sample well, 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 20 times concentrated washing liquid with distilled water 20 times and 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 at 37 ° C in the dark for 15 minutes.
10. Termination: Add 50μl 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.
Summary of operating procedures:
1. Prepare reagents, samples and standards;
2. Add the prepared samples and standards, and react at 37 ℃ for 30 minutes;
3. Wash the plate 5 times, add enzyme reagent, and react at 37 ℃ for 30 minutes;
4. Wash the plate 5 times, add color developing solutions A and B, and react at 37 ℃ for 10 minutes;
5. Add stop solution;
6. OD value within 15 minutes;
7. Calculation.
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