Mysterious case of IgG precipitation: Imidazole+glycine+freezing = insoluble ppt ???

Dima Klenchin klenchin at
Wed Sep 13 21:30:15 EST 2000

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):

- thawing at 37C or on ice;
- adding NaCl to 0.1-1.0 M;
- diluting 10X with PBS/HBS/TBS
- diluting 10X with either alkaline or acidic solutions,
with or without salt (9.0 or 2.5).
- it does dissolve readily in 1% SDS. 

OK, is there something magic about imidazole/glycine
buffers and the freezing that affect all proteins? As a control
for this, I diluted two concentrated protein samples (rabbit 
serum and rat brain cytosol) 10X with the exact same mixture
of glycine and imidazole and froze the samples. NO, there 
was absolutely no any precipitate upon thawing. 

Thus, it appears there is something fairly specific about 
freezing IgG in this buffer at high enough concentration 
(another prep, prepared same way but at 1.2 mg/ml shows
only ~25% of total protein precipitated upon thawing, the rest
is soluble and active).

What is an explanation? Help, I am losing all the self-respect
I've ever had! :-))

        - Dima

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