Several studies have shown that washing at 4 degrees Celsius can improve the specific signal for western blotting. For example, one study found that washing at 4 degrees Celsius increased the specific signal by up to 50%. https://precisionbiosystems.com/wp-content/uploads/2019/08/mayo-app-note-blotcycler.pdf

Another study found that washing at 4 degrees Celsius reduced the background signal by up to 80%.

Washing at 4 degrees Celsius can help to reduce non-specific binding by slowing down the diffusion of the primary antibody. This gives the antibody more time to bind to its target protein and less time to bind non-specifically. This can also improve the specific signal for western blotting; lower temperatures can help to reduce non-specific binding of the primary antibody to the membrane.

Non-specific binding can occur when the primary antibody binds to proteins on the membrane that are not its target protein. This can lead to false positive bands on the western blot.

In addition, washing at 4 degrees Celsius can help to reduce background signal. Background signal is caused by unbound primary antibody and other proteins in the washing buffer. By washing at a lower temperature, the background signal can be reduced, making the specific bands more visible.

Overall, washing at 4 degrees Celsius is a good way to improve the specific signal and reduce the background signal for western blotting.

Here are a few suggestions for washing western blot membranes at 4 degrees Celsius:

  • Run all blot processing steps at 4 degrees C.
  • Use a washing buffer that is pre-chilled to 4 degrees Celsius.
  • Proper Agitation of the membrane during the washing steps.
  • For the best results, use a system that automates all the blot processing steps. The BlotCycler delivers precise timing of each step(blocking, primary/secondary antibody, washing) delivering reproducible and publication quality results.