Purification of antibody raised against hexahistidine tagged protein

Leo 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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