First off thanks for taking a look and helping me out. So I transferred my protein to a nitrocellulose membrane a couple of days ago. Initially, I was able to visualize my protein, however, there was some splotchy non specific reactivity at the top of the blot which I attributed to some sort of contamination in my milk. I stripped my membrane for a little over an hour and blocked with 5% milk and put my new antibody on overnight. This was an older antibody which I had used several times. I incubated at 4 degrees overnight, and when I pulled my blot the next morning the primary antibody containing milk smelled funky. I tried to visualize anyways and I was unable to see anything. I then tried an excellent primary antibody that I have and I still cannot visualize anything. Is it possible that I have uniform bacterial contamination on my blot which is block all antibody binding sites? Is there anything I can do to save this blot?
I am blocking with 5% milk and washing with PBS.
Thanks a lot for any suggestions