IgM for affinity chromatography

GARBRECHT fgarbrec at POST.ITS.MCW.EDU
Sat Dec 19 12:33:22 EST 1992


On 11 Dec 1992, NGUYENHD wrote:

> Date: 11 Dec 1992 15:14:22 -0500
> From: NGUYENHD <nguyenhd at hermes.bc.edu>
> To: Methods and Reagents <Methods at net.bio.net>
> Subject: IgM for affinity chromatography
> 
> I have purified IgM antibody from my culture media by Sephacryl 300.  I have
> also tried to couple this antibody to CNBr-activated Sepharose 4B.  Antibody
> binds to the beads, but it loses its activiy and leaks with the eluting buffer
> (0.1M Glycine, pH 2.5).  How do I get around these problems?
> 
> Thank you in advance.
> 
> Hung Nguyen
> Biology Dept., Boston College
> NGUYENHD at hermes.bc.edu

If your antibody leaks off after coupling to CNBr activated beads, then your
coupling was not complete and you are washing off unbound antibody.  Wash the
column more after coupling to make sure all unbound material is freed from
the column before you use it.  Also, some antibodies are quite sensitive
to low pH.  Try successiviely higher pH (2.5, 3.0, 3.5, 4.0, 4.5) to see
whether that will work without denaturing the antibody.  You may also have
better results at high pH (10 or 11).
Fred Garbrecht
Medical College of Wisconsin
fgarbrec at post.its.mcw.edu





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