What should I do if I have a high background (uniform distribution) in western blot?

There are various solutions to resolving high backgrounds in western blot. One solution is to change the conditions that prevent interference by incorporating a blocking substance into the antibody solutions. Another solution is to experiment with different amounts of the antibody to determine the optimal concentration (titration). One can also gradually reduce the quantity of protein loaded in each lane to prevent overloading. Increasing the number of wash cycles during the washing steps of the blotting process to help remove unbound antibodies. The blot should also be kept at a low temperature (typically around 4°C) during incubation steps. Using a nitrocellulose membrane instead of polyvinylidene difluoride (PVDF) is another way to resolve a high background issue in western blot. Instead of using milk as a blocking reagent, one can consider using bovine serum albumin (BSA). Do not let the membrane dry out during processing and incubation steps. Lastly, including a gentle detergent like Tween 20 in the solutions used for incubating and washing the blot, particularly when working with antibodies that target phosphoproteins.