Experimental Step 1. Encapsulated Antibody
1) Dilute the capture antibody to a concentration of 1-10 μg / ml in carbonate / bicarbonate buffer (pH 7.4) and coat the microtiter plate.
2) Cover the microplate with parafilm and incubate at 4 ° C overnight.
3) Discard the coating solution and wash the plate twice with phosphorus PBS, 200 μl per well. Gently shake off the lotion on the sink, and pat the enzyme plate on a paper towel to remove residual liquid.
2. Close and add samples
1) Block the remaining protein binding sites in the wells after coating, and add 200 μl of PBS containing 5% skimmed milk powder to each well.
2) Cover the microplate with parafilm and incubate for at least 1-2 hours at room temperature, or 4 ° C overnight if convenient.
3) Add 100 μl of appropriately diluted sample to each well. In the accurate quantitative determination, the samples of unknown concentration are usually compared with the standard curve, and each plate should be made a standard (two or three copies) and a blank control to ensure accuracy. Incubate at 37 ° C for 90 minutes.
4) Discard the sample and wash the plate twice with PBS, using 200 μl per well.
3. Incubate with detection antibody and secondary antibody successively
1) Add 100 μl of diluted detection antibody to each well.
2) Cover the enzyme plate with parafilm and incubate at room temperature for 2 hours.
3) Wash the plate 4 times with PBS.
4) Add labeled secondary antibody, 100 μl per well, and quickly dilute to the best concentration with blocking solution before use (refer to the instruction manual).
5) Cover the enzyme plate with parafilm and incubate for 1-2 hours at room temperature.
6) Wash the plate 4 times with PBS.
4. Detection
Add the substrate solution using a multi-channel pipette, 100 μl (or 50 μl) per well.
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