Just a thought. Have you tried dissolving it in water insted of PBS?
Allen Black
medm003 at uabdpo.dpo.uab.edu
Dept. of Microbiology
Univ. of Alabama at Birmingham
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.
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