The antigen-antibody interaction primarily depends on the specific activity and specificity of the antibody against a given antigen and the amount of antigen present in the sample. However, there are certain experimental factors that can affect the results. These include any changes in concentration of the antigen, antibody or substrate. So every time any one of the variables is changed there is need for optimization.
The most commonly used procedure to determine the optimal antigen and antibody concentrations is to perform a western blot with varying dilutions of the antigen and antibody. A more simpler and quicker way to determine the optimal antigen-antibody concentration is to perform a dot blot.
Further, if you observe any of these results when you do a western blot - it is time to optimize your antigen-antibody concentrations!
- Nonspecific bands
- No signal with no background
- Signal fades quickly
- Bands are diffused
- Too much background
