I have sucessfully precipitated an IgM monoclonal ab from one liter of
serum free tissue culture sup using a 25% PEG cut. This culture sup (Gibco
SFM) contains my IgM, along with transferin and insulin which are initialy
contained in the media at 205g/ml. I intend to further purify out the IgM
(900kd)from the transferin (~80kd) and insulin (~6kd) by size exclusion
chromatography on a Sephadex G-100 column. My current problem is that I
can't get the IgM containing precipitate from the PEG cut into solution in
room temp PBS (10 mls). I can get it suspended, but it comes right out of
suspension. Even if all of the transferin and insulin came down in the PEG
cut, the resulting 20 mg of protein (from one liter) should easily go into
solution in 10 mls of PBS, right? Something must be up with the IgM? Do you
have any ideas for getting my IgM containing precipitate into solution so I
can continue with the purification? Perhaps other solvents? Thanks for
your help. If you are interrested in the serum free media, let me know, I
had great luck getting my clones to grow in this stuff.
thanks for all your help
--
Jonathan Kurtis
International Health Institute
Brown University Box G-8033
Providence, RI 02912
