Western Blot Help - (Aug/22/2014 )

Hello all, 

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

Morgan

It is more likely that the bacteria have secreted proteases which have digested the proteins on the blot. Unfortunately you will have to repeat.