I have been having issues with my Streptavidin-Cy5 blots. When I run the SDS-PAGE, electrotransfer, and blot with SA-Cy5, random bands go across the entire gel, even across empty lanes. I have attached images of the issue. The first image is Cy5 visualization (there is a biotin probe and I expect signal up each of three lanes). The second image is the other half of the same gel stained for sypro. The third image is a separate gel made from the same components that is a PKA WB (the white band is where it's supposed to be). Note that in the SA-Cy5 blot, a shadow appears in the top half of the gel only.

I have remade and double checked the composition and pH of all of my gel components and my electrotransfer buffers. I have used different power supplies, electrodes, all the hardware. When I use the same materials to western blot a protein, it looks fine. When I use the same materials to stain for total protein with Sypro RUBY, it looks fine. The ladder on the problem blot runs normally and transfers normally. Proteins migrate to their correct sizes (checked by WB and visualization with HRP). I have used both SA-Cy5 and SA-HRP and the result is the same. I have also used different imagers and exposure settings to take the pictures. The attached images are representative - none of these fixes has corrected the problem.

The problem is streptavidin-specific, and specific to me. My colleagues have used the same SA-Cy5 stain and their blots look normal. Many of the components of gel making are shared throughout the lab, and no one else has reported any issue. Those components that are not shared I have remade twice, triple checking the recipes against 3 different sources. I have been dealing with this issue for over a month, and I'm at the end of my rope. Does anyone have any ideas?