I recently did western blots with Pbp2a polyclonal antibodies to detect the presence of protein of interest–penicillin binding protein 2a (Pbp2a). The expected product size is ~75kDa. Goat anti-rabbit peroxidase is my secondary. The following are the problems I encountered.
1. Consistently, I get another band below ~50kb. Sometimes this band is much stronger than the band I’m probing for. Anyone has any ideas as to what this band is??
2. I did a control (strain known to not express mecA, the gene that encodes Pbp2a), but I still see a very faint band. Has anyone see this with their westerns?
Anyone who had done work with Pbp2a, please respond. Thanks!
I don’t know if you are still working on this, but one possiblity is that your protein is phosphorylated. You would see two bands, one phosp. and one de-phosph, which fits your discription. A way to check for that is to treat with CIP (calf intestinal phosphotase) and see if one of the bands disappears. There are also web data bases that will take a protein sequence and annotate likely positions that would be phosphorylated (search Google or similar, can’t remember web pages at the moment) if you needed to do further research. I am assuming the background is low to non-existent on your membrane. If you do have background, of course it could be non-specific binding which would require annoying amounts of work to figure out the best conditions (sometimes a secondary antibody raised in a different animal will give you different results). Hope this helped a little!
I just re-read your post and realized what I wrote is not necessarily applicable! (Since you ran the control – as long as you trust those results) Trying a different secondary might still be an option however.