Immunoaffinity Purification Using An IgM
droberts at joplin.biosci.arizona.edu
Mon May 9 18:50:01 EST 1994
In article <udbl119.15.000E2B2B at bay.cc.kcl.ac.uk> udbl119 at bay.cc.kcl.ac.uk (Mandy Johnstone) writes:
>I have a monoclonal antibody which is an IgM and I would like to use it to
>purify the protein that it recognises. I think the best way to do
>this is by doing an immunoaffinity isolation, linking my antibody to a solid
>support column but I do not know how effective it will be with an IgM antibody
>as apposed to an IgG. I was wondering if anyone out there has had any
>experience linking IgM abs to a column and the best way to go about this.
>Also is there a commercially available kit that I can buy that you would
>recommend. I have an abundant supply of the monoclonal. Many thanks in
>(M.Johnstone at bay.cc.kcl.ac.uk)
>King's College, London.
Well, there are several ways to go about it, most of which that I'm
aware of involve hooking up the Antibody to an activated stationary phase via
its N-terminus (or a lysine NH3 group.) You can buy many resins that are
already activated from Sigma, or you can activate them yourself.
I did this several years ago using cyanogen bromide activated agarose
and it was somewhat of a pain. It was inefficient in my hands, so you may
want to buy it, although I'll bet it will cost you.
The purification worked well, but you have to worry about denaturing
your protein when you elute it.
We used pH 2.3 to elute, and eluted directly into tubes containing
some ungodly buffer concentration at pH 7.0.
I have a general reference for Affinity purification, and if I
remember, there is a section on preparing columns and elution.
Methods in Enzymology, Vol. 104, 3-68, 1984.
One more thing: If you decide to activate the matrix yourself, using
cyanogen bromide, don't let the pH get acidic, as cyanide gas is released.
(i.e. do this in the hood).
You might consider other activated matrices like tresyl, epoxy,
carbonylimidazole, or succinamide. I've heard that they work well, but have
never used them.
Good luck, Doug R.
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