I am immunizing rainbow trout with bacterins (crude bacterial protein preps) from a potential pathogen. Running the bacterins on a SDS-PAGE gel shows a fairly uniform distribution of proteins throughout the normal molecular weight range for a 12% gel.
We immunize fish with these bacterins and after about a month collect blood and then isolate the serum.
I then use these sera to probe Western immunoblots of the bacterins and visualize using an mouse anti-rainbow trout H chain-MAb and a goat anti-mouse polyclonal-HRP. The 2nd and 3rd antibodies were checked for cross reactions to the bacterin and each other.
What I am seeing is a strong reaction with many bands in the molecular weight region above 60kDa, almost nothing below 60kDa except a few isolated bands around 25kDa.
Ponceau red staining of the membrane shows that the proteins in below 60kDa are transferring properly. Bacterins of other species of bacteria that were not used for immunization were run on the same gel, and there was no signal, so the above 60kDa signal seen with the immunized bacterin is specific, but not necessarily antibody mediated.
Why is there a big hole with no signal in the Western Blot below 60kDa? I attended a seminar of a researcher doing the same thing in totally different bacteria and host and saw the same pattern. Not much below 60kDa.
Bigger proteins have more antigenic potential so it is not surprising to see a lot of signal there, but it seems what I am seeing is am methodology problem.
Any advice?
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