Antibody Affinity Purification

John Leonard via methods%40net.bio.net (by jleonard.iii from gmail.com)
Thu Jun 19 17:26:03 EST 2008


Hello all,

I have serum containing a custom polyclonal antibody and I'd like to
affinity purify it via interaction with its ligand.  I have His-tagged
antigen that I would like to immobilize on a Ni-NTA column.  I would, then,
run diluted serum over this column to pull out any antibodies specific to my
antigen, then selectively elute the antibody (ex. with high salt).  I know
this is possible and have an idea for a protocol, but I was wondering if any
of you have done this before and have any pointers or protocols that have
worked well for you (i.e. specific buffers, amounts of antibody and antigen
to use, etc).

Any suggestions are welcome!  Thanks!


John


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