I have been running western blots to examine protein binding to ribosomes and noticed something interesting. When I run my protein of interest alone in a lane, I am able to successfully detect it by western blot using either my antibody (serum raised against my POI), or a commercial anti-5xHis antibody. However, when I add purified ribosomes to my protein of interest and run this mixture on a PAGE gel and western blot, the signal from my protein of interest either disappears or decreases heavily. I am able to run the ribosomes on their own successfully and have verified their entry into the gel via coomassie stain. This is a new phenomenon which occurred only in the last couple of weeks, as before this I have been able to detect my protein without issue. There is BME in all my loading dye and the dye it self is a 4x concentration used at the correct dilution (to 1x) in the gel sample.
Does any one have any idea why this is happening?