Eluting triple tagged protein from antibodies?

DTerBush danter1 at aol.com
Fri Mar 21 12:39:22 EST 1997


Has anyone ever been successful eluting a triple c-myc tagged protein
from antibody using the 9E10 peptide? I recently tried this trick and
failed. I tried adding 125 µg of 9E10 peptide in 0.5 ml of buffer in an
attempt to elute a triple-c-myc tagged protein from, what I estimate to
be about 2 ug of total 9E10 antibody immobilized on 4 mg (dry weight) of
protein A beads (yes I know this monoclonal is supposed to bind protein
G better, but I can't see any difference under my experimental
conditions), and I see almost no elution after incubating overnight at
4C. After thinking about this, it might not be possible to elute the
protein since the antibody is divalent and can bind to two of the three
c-myc epitopes on the protein simultaneously. Thus, even if you
transiently broke one of the two connections to the antibody, the
protein won't float away. Since it would stay localized close to the
other two unoccupied c-myc epitopes on the protein, the effective
protein-c-myc concentration must be high leading to a very high
likelyhood of rebinding the protein-c-myc before the other
protein-c-myc-antibody interaction could be competed off by free
peptide.



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