Immunochemistry question (probably dumb)

Peter Ellis via methods%40net.bio.net (by pjie2 from cam.ac.uk)
Mon Jun 20 14:09:23 EST 2011


Can you use a goat anti-rabbit secondary and a rabbit anti-goat 
secondary in the same reaction?  Every fibre of my being says no - 
they'd either precipitate out or at the very least give nonspecific 
signals.  On the other hand, I'm reading a paper that claims to have 
done just that.

To reiterate, they are trying to detect two proteins, A and B.  The 
primary ABs are rabbit anti-A and goat anti-B. The secondary ABs are 
Cy5-[goat anti-rabbit IgG] and FITC-[rabbit anti-goat IgG] conjugates. 
Surely these two secondaries should mutually detect each other and 
obliterate any signal from the underlying A and B?  They're claiming a 
perfect non-overlap of signals, with 50% of cells containing A and 50% 
containing B.

Someone here is missing something, and I really hope it's not me.

Peter Ellis.


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