1. Coating
(1) Dispense 200ul (may vary from 50 to 200ul depending on
user's need) of prepared Coating Solution to each well.
(2) Incubate 1 hour at 37°C. (or 1-3 hours at room temperature/
overnight at 4°C)
2. Washing (All washing method is the same.)
(1) Remove the solution from each well and fill up the Washing
Solution. Repeat 3-5 times. Complete removal of liquid in each
step is essential to good performance.
(2) After the last wash, remove any remaining Washing Solution.
Turn over the plate and blot carefully on paper towels.
3. Blocking
(1) Add 250ul Blocking Solution to each well.
(2) Incubate 1 hour at room temperature or at 37°C.
4. Washing
5. React Sample/Standard (or Primary Antibody)
(1) Add 200ul diluted Sample/Standard (or Primary Antibody) to
each well.
(2) Incubate 1 hour at room temperature or at 37°C.
6. Washing
7. Add HRP-conjugated Detection Antibody (or Secondary
Antibody)
(1) Add 200ul diluted Detection Antibody to each well.
(2) Incubate 1 hour at room temperature or at 37°C.
8. Washing
9. Color Reaction and Reading
(1) Dispense 100ul of pink-ONE TMB to each well.
(2) After sufficient color development (5-10minutes at room
temperature or at 37°C), add 100 ul Stop Solution (2M H2SO4) to
each well.
(3) Using a microtiter plate reader, read the plate at the
wavelength that is appropriate for the substrate used
(450 nm for TMB).
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