achimr at home.com
Tue Aug 24 21:57:09 EST 1999
Does it have cysteines? If yes, you have to check whether your protein
might have intramolecular SS-bridges.
Dialyze against a 10:1 ratio of reduced : oxidized glutathione.
Rajesh Kumar <rk6n at virginia.edu> wrote in message
news:37C08DFC.8250BE29 at virginia.edu...
> I have expressed a new protein in BL-21 cells using pET-28 vector. I
> could purify it under denaturing (in presence of urea) condition using
> HIS tag. The problem is that as soon as I try to get rid of urea by
> dialysis (against PBS) or ultrafilteration the protein comes out of the
> solution. I tried to dissolve the precipitates in detergents (triton
> X-100, Tween-20), low pH buffer, high pH buffer but without success.
> On the basis of amino acid composition the protein (18 Kd) is slightly
> on the hydrophillic side except a 20 aa signal peptide region. Its
> predicted pI is ~ 9.7 (The pI of the protein itself is ~5.3 but the
> extra amino-acids that come from the vector make it 9.7).
> I will appreciate if somebody could suggest me a way to make the
> protein soluble in a system that doesn't interfere in most protein
> analysis work.
> rk6n at virginia.edu
> University of Virginia, Charlottesville .
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