In article <Jonathan_Kurtis-181093231318 at tonto-slip2.cis.brown.edu>,
Jonathan_Kurtis at Brown.edu (Jonathan Kurtis) wrote:
>> 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 20g/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.
Well, I usually use Ammonium Sulphate precipitation for such things. I
would
try DMSO to dissolve the IgM/PEG, or try to put the goop into 25% peg, and
then with stirring, gradually add water drop by drop, to see if gradually
decreasing the concentration of peg works better than rapid change. I
presume the PEG is not dialyzable. If you get something to work, please
post it. The experience will be good to know about.
Steve Holland
