Mysterious case of IgG precipitation: Imidazole+glycine+freezing =
insoluble ppt ???
hzhen at freeuk.com
Sat Oct 7 11:27:01 EST 2000
Dima Klenchin wrote:
> I am pulling all my hairs out over it!!! Never came accross
> something so seemingly simple yet something I have no
> idea how to explain. This is not about science or my ruined
> prep anymore. I just want to *understand*!
> I purified some rabbit IgG on Protein A column. Elution
> was with 50 mM Glycine pH 2.5. Extremely clean and efficient.
> Because Tris is highly inibitory for the assay I was going to
> use these IgG for, I decided to neutralize with harmless base,
> imidazole (to avoid dialysis and sample loss).
> 100 ul 2.0 M Imidazole, pH 9.0 + 1 ml 50 mM Gly, pH 2.5 =
> = pH ~ 7.1. The final prep was perfectly clear solution of
> 2.8 mg/ml IgG. Then I aliqioted and froze the thing
> (150 ul in eppies stuck in -80C)
> Upon thawing, huge proteinous ppt formed! Over 90% of
> the total protein was not in solution anymore. Since nothing
> I know suggest that this might happen, and since the prep
> is quite valuable (serum source is limited), I started little
> investigation hoping to get the IgG pellet back into solution
> (nothing worked):
Protein precipitating on freeze/thaw is not an unknown problem, and
has little to do with the buffer I think. If you want to freeze
protein, you should always snap-freeze in liquid nitrogen before
putting them into freezer. There is the phenomenon of cold
denaturation in which protein becomes denatured at low temperature
which could well leads to aggregation, although I can't say if this is
the case with your protein.
> - Dima
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