Purification of antibody raised against hexahistidine tagged protein
klebs.bbs at bbs.life.nthu.edu.tw
Sun Apr 28 09:55:13 EST 1996
It seems that more and more laboratories are using HisTag
as a fusion partner when expressed a foreign gene in E. coli.
Frequently, the recombinant proteins are used to raise
antibodies in rabbits. The most common way to purify the antibody
specific to the protein of interest is to cross-link the antigen
to CNBr activated CL4B beads, absorb the specific antibody, then
elute the antibody by a somewhat rough reagent. In some cases, the
eluted antibody may lose activity irreversibly.
Since the HisTag fused protein binds to the Ni++ charged resin
readily, the antigen-bound column may be used for the purification of
the antigen specific antibody. Specifically, I am interested in
knowing that if there is a reliable reagent which is strong enough to
elute the antibody from the column but is gentle enough not to
coelute the antigen. Of course, the antibody should also be stable
under such a condition.
Thank you very much in advance for sharing with me your experience.
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