Hi Lee,

Possibly a bubble may have made the gel not perfectly in contact with the membrane. To avoid bubbles, you can pass a tube by gently pressing on the gel from one end to the other, to ensure it contacts the membrane evenly.

Dear María Luisa Caballero Thank you for your comment，I removed air bubbles between the gel and membrane carefully every times and the diffuse bands would only appear at the middle of the membrane

maybe make the filter paper sandwich tighter fitting? so if doing semi-dry for instance, add an extra 1-2 filter papers on either side of the gel/membrane. And yes, roll out bubbles definetly.

The appearance of diffuse bands on the membrane after a Western transfer could indicate issues with protein aggregation, improper gel handling or loading, or insufficient transfer efficiency.