What roles do SDS and alcohol play in the western blot transfer step?

In the transfer step of Western blotting, SDS facilitates the release of proteins from the gel, particularly when certain proteins are challenging to extract. Specifically, SDS helps to denature proteins by disrupting non-covalent interactions. Negatively charged SDS molecules attached to proteins facilitate the movement of protein complexes towards the positive pole (anode) during electrophoresis. This separation by size enables the differentiation of proteins based on their molecular weights. However, the presence of SDS in the transfer buffer reduces the effectiveness of protein binding to the nitrocellulose membrane.

Alcohol, such as methanol, isopropanol, or ethanol, is often added to the transfer buffer to enhance the binding capacity of nitrocellulose membranes for proteins during Western blotting. It does this by increasing protein hydration. Alcohol also serves to remove SDS from proteins that have been transferred from denaturing SDS-containing polyacrylamide gels. Additionally, ethanol helps stabilize the gel's structure during the transfer process and contributes to the formation of a uniform electric field in the gel, ensuring even transfer of proteins across the membrane.