Coating 96-well microplates with protein for ELISA
Assay of proteins with a protein specific antibody
- Add 200 µl of anti-target–protein antibody diluted 1/2000 in PBS/BSA. Cover plate, and incubate for 1–2 h at RT.
For higher sensitivity, antibody binding can be performed overnight at 4°C.
- Wash wells 4 times with PBS-Tween. Soak wells for 10–60 s per wash, and dry the wells by gently tapping the plate on paper towels after the wash.
- Dilute secondary antibody in PBS/BSA according to the manufacturer’s recommendations. Add 200 µl of the diluted antibody to each well, and incubate at room temperature for 45 min.
- Wash wells 4 times with PBS-Tween. Soak wells for 10–60 s per wash, and dry the wells by gently tapping the plate on paper towels.
- Add 200 µl of substrate solution, and monitor color development in a microplate reader.
Note: Substrate solution should always be prepared immediately before use.
Tip: Monitor color development over a period of 45 min, or add 50 µl stopping reagent after a specific time and measure product. When testing a new assay system, a time-course of color development should be carried out to determine optimal development time and temperature.
Tip: If the reaction is stopped the signal will increase slightly, depending on the substrate used, and the color will be stable for a certain period of time.