! Antibody purification

Ron Smits ron at ruly46.medfac.leidenuniv.nl
Mon Nov 20 12:34:55 EST 1995


We have raised several rabbit polyclonal antibodies against fusion proteins of approximately 300
aminoacids. All of them recognize on westerns the expected protein but as expected for polyclonals,
also a lot of extra bands (specific aspecific).
When we tried  to get rid of all these extra bands with affinity columns, the pictures on western were
cleaner with respect to background but not for the aspecific bands. Does anyone know why we
can't get rid of these aspecific bands? Is there a protocol for purification which has more chance of
success?
We have thought ourselves of using proteinextracts of tissue which doesn't contain the protein of
interest, to compete out the aspecific antibodies. Some people told us this does work in principle but
not in practice. Is there anyone who has some success stories on this matter?
Any suggestion is welcome!

Ron Smits
Leiden University




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