On 3 Dec 1992, NGUYENHD wrote:
> Date: 3 Dec 1992 19:54:14 -0500
> From: NGUYENHD <nguyenhd at hermes.bc.edu>
> To: Immunology <Immuno at net.bio.net>
> Subject: Purify IgM
>> Does anyone have a protocol to purify monoclonal antibodies IgM? I have tried
> Protein A agarose affinity column with no success.
> If you have a protocol, please reply to
>NGUYLENHD at hermes.bc.edu (Hung T. Nguyen)
As you have discovered, IgM does not bind to Protein A, and thus cannot
successfully be purified that way. We purify IgM using a 2 step method:
first precipitate the IgM by dialyzing against cold distilled water (at least
2 changes over 24 hours); some IgM antibodies will precipitate out at
low ionic strength, but not all. If your antibody does not, then you should
precipitate it by adding an equal volume of saturated ammonium sulfate.
Collect the precipitate and dissolve it in the buffer of your choice
(we use PBS). The preparation may be pure enough at this stage, but
you will probably have some contaminating transferrin and albumin. If you
need to purify it further, it is a simple matter to run it over a size
exclusion column (Sephacryl S300); IgM with a MW of 900,000 is much larger
than most other contaminating proteins, so you should end up with a
reasonably uncontaminated preparation. Good luck,
Fred Garbrecht
Medical College of Wisconsin
fgarbrec at post.its.mcw.edu
