Secondary antibodies are typically polyclonal antibodies which means that they are made up of a mixture of different antibodies that recognize different epitopes on the target protein. This can therefore lead to non-specific binding to various proteins during Western blotting. Some of these antibodies may also recognize epitopes on other proteins, besides your protein of interest.

Factors to consider that can contribute to non-specific binding of secondary antibodies, including:

  • Incomplete blocking can lead to more free binding sites on the membrane for the secondary antibodies to bind to, which can increase the probability of non-specific binding.
  • Too high a concentration of secondary antibodies can also increase the chances of non-specific binding. This is due to more antibodies available to bind to the membrane, even if only a small percentage of them recognize epitopes on the target protein.
  • If your sample is in low protein concentration, there will be fewer target proteins for the secondary antibodies to bind to. This can increase the probability of non-specific binding, as the secondary antibodies will have more free binding sites to bind to.

Here are a few suggestions that can be done to reduce non-specific binding of secondary antibodies:

  • Assess the quality of the secondary antibody by performing a blot without primary antibody.
  • Perform a titration experiment with the antibody to determine minimal background and maximum signal. With the Blotcycler touch up to six different secondary antibody titrations can be run simultaneously; optimization can be accomplished in one run.
  • Performing thorough blocking and considering dilution in a stronger blocking agent that yields less background. Incubating the membrane with a blocking solution for an extended period (typically 1-2 hours).
  • Lower the concentration of secondary antibodies. The recommended concentration of secondary antibodies can vary depending on the manufacturer, typically a concentration of 1:10,000-1:100,000 is sufficient.
  • Using a higher protein concentration in the sample will help to ensure that there are enough target proteins for the secondary antibodies to bind to.

Automate the Blot Processing step https://www.blotcycler.com/