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Why can't we detect multiple protein in a single membrane using western blot - (Aug/07/2012 )

I am trying to detect three proteins A, B and C. Unlike Immunohistochemistry, Why i cannot detect all the proteins in a single membrane when the antibodies against my protein are raised in different animals? I know the size of my proteins.


Some people do this checking their protein of interest plus a loading control, for example, specially when they know perfectly well how their antibodies are working on their samples and on their reagents (blocking and washing buffer, antibody concentrations, type of membrane...). The problem is that many antibodies have non-specific bands that might complicate things... . At the end, it is easier and more convinient to run different western blot at the same time or stripping the membrane if sample is difficult to obtain.


I have routinely run up to 4 different antibodies on the same blot at the same time (and different times) with no problems. You have to have carefully optimised the concentrations of the antibodies and determine if the secondaries are likely to bind more than one target or not.