[Protein-analysis] Re: Protein Solubility Problem

J Rice rice.jeffrey at gmail.com
Sun Aug 13 09:06:01 EST 2006


DK wrote:
> In article <1155311077.292056.91660 at 74g2000cwt.googlegroups.com>, rice.jeffrey at gmail.com wrote:
> IMHO, two possibilities:
> 1. Your protein denatures irreversibly in PBS and requires 0.5 M to
> stay happy. This is a bit of a stretch but possible. The solution is to
> dialyze agains phosphate-buffered 0.5 M salt.
> 2. Your protein is insoluble in the presence of Ni2+. I actually had
> such a case - precipitate upon dialysis, won't redissolve in high
> salt. However, it did redissolve upon addition back of imidazole.
> This was a hint. So, I tried 2 mM EDTA and it went back into
> solution in low salt quite nicely. (There is *always* some Me2+
> leaching out during IMAC).

I think your first point is probably correct -- not that the protein is
natively insoluble in PBS, but that the rapid change from 0.5M to .14M
during dialysis caused the problem.  As my lysate is already in 0.5M, I
will dialyze against PBS w/ 0.5M NaCl, but also consider performing all
the steps at lower NaCl in the future.

Thanks.



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