Biochemical Techniques and Instrumentation

Procedure

Coating antigen to microplate

1. Dilute the antigen to a final concentration of 20 μg/ml in PBS or other carbonate buffer.
2. Coat the wells of a PVC microtiter plate with the antigen by pipeting 50 μl of the antigen dilution in the top wells of the plate.
3. Dilute down the plate as required.
   • Test samples containing pure antigen are usually pipeted onto the plate at less than 2ug/ml.
   • Pure solutions are not essential, but as a guideline, over 3% of the protein in the test sample should be the target protein. (antigen).
   • Antigen protein concentration should not be over 20ug/ml as this will saturate most of the available sites on the microtitre plate.
   • Ensure the samples contain the antigen at a concentration that is within the detection range of the antibody.
   Blocking
4. Block the remaining protein-binding sites in the coated wells by adding 200 μl blocking buffer, 5% non fat dry milk/PBS, per well. Alternative blocking reagents include BlockACE or BSA.
5. Cover the plate with an adhesive plastic and incubate for at least 2 h at room temperature or, if more convenient, overnight at 4°C.
6. Wash the plate twice with PBS.

Incubation with Primary and Secondary antibody

1. Add 100 μl of diluted primary antibody to each well.
2. Cover the plate with an adhesive plastic and incubate for 2 h at room temperature. This incubation time may require optimisation. Although 2 h is usually enough to obtain a strong signal, if a weak signal is obtained, stronger staining will often observed when incubated overnight at 40^oC.
3. Wash the plate four times with PBS.
4. Add 100 μl of conjugated secondary antibody, diluted at the optimal concentration in blocking buffer immediately before use.
5. Cover the plate with an adhesive plastic and incubate for 1-2 h at room temperature.
6. Wash the plate four times with PBS.