Dr M.R. Clark
mrc7 at cus.cam.ac.uk
Mon Aug 8 11:46:21 EST 1994
In article <30n6fj$m3m at bigfoot.wustl.edu>, scott gens <gens at biodec> wrote:
>I have a 200 uL of a monoclonal mouse IgM antibody in ascites fluid that I
>would like to purify. This is all I have so I need to get it right on the
>Protein A supposedly binds to IgG and IgM, but most catalogs present
>tables which state that their Protein A columns do not bind mouse IgM. Is
>mouse an exception, then? Why the apparent contradiction?
>BioRad's catalog seems to suggest that their Protein A does bind mouse IgM
>but they insinuate that this only happens when their 'MAPS buffer' is used.
>Then they also state that 'approximately 50% of all mouse IgMs bind to
>protein A'. So does this mean that basically I only have a 50% chance of
>purifying my IgM using their system, and if so, why should I want to gamble
>on my IgM being in the lucky 50%, especially when none of the other catalogs
>I have make similar claims?
The reason for the apparent varaibility is that some V-regions can
bind to protein A so some antibodies of all isotypes will bind.
Probably the best way to purify IgM is by gel filtration, it's very
high molecular weight allows a reasonable seperation from other serum proteins
including IgG. I've used Aca22 for this in the past.
Alternatively IgMs are euglobulins and will precipitate at low ionic
strength so you could dialyse against say 10mM phosphate buffer or even
water! The only problem I've had with thi approach is that it can prove
difficult to redissolve the pptte!
Good luck :-)
More information about the Immuno