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Western blot background and specificity - How good is my antiserum? (Nov/04/2005 )


I had polyclonal antisera raised against my favorite protein. In western blots there is a specific band plus a significant amount of schmutz. In IHC there is a clean signal in appropriate tissues, and no signal with pre-immune serum. The pathologist reading the IHC believes the signal, the technician who made and tested the antibody believes it at least sees its target protein, but admits there may be some background. A manuscript using this antibody is being prepared. Do I need to kill myself to prove specificity by Western? Or is the existing data sufficient. I am used to DNA and RNA where I can always get squeaky clean results.


It really depends on the reviewers and what caliber journal you are sending it to. I just finished reading a paper for journal club where it was pretty clear the reviewers made them use RNAi to the protein and show loss of IHC staining to prove specificity.

If you're lucky you may get away with not doing it, but personally, after working with a supervisor who often skimps on control reactions for a long time, I would do the control reactions. But then again I've had bad luck with antibodies. I've gotten burned before, wasting several months on bad vendor antibodies. Usually when the right mol. weight light up on western the antibody is good, but not always.

It's really your decision, Good luck