Mysterious case of IgG precipitation:
Imidazole+glycine+freezing = insoluble ppt ???
John E. Wiktorowicz, Ph.D.
johnw at lynxgen.com
Wed Oct 11 14:38:34 EST 2000
The nice thing about most IgG's is that they can undergo denaturation, and
renature spontaneously. Try solubilizing in a chaotrope like potassium
thiocyanate (~2M) and then dialyze it away. Guanidinium may alos work.
> From: ChenHA <hzhen at freeuk.com>
> Newsgroups: bionet.molbio.methds-reagnts,bionet.molbio.proteins
> Date: Sat, 07 Oct 2000 17:27:01 +0100
> Subject: Re: Mysterious case of IgG precipitation: Imidazole+glycine+freezing
> = insoluble ppt ???
> 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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