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

Jan Leunissen leunissen at aurion.nl
Fri Oct 13 10:29:11 EST 2000

I am not surprised. We have the same. I don't know if what I am going to
write is completely correct, but this is what I think happens:

Many proteins precipitate when they are at their IEP. Many rabbit IgG's
have an IEP of about 7. And on top of that, rabbit IgG is relatively
hydrophobic in its Fc part (that's probably why SDS helps). That makes
them easy to precipitate, much easier than for instance goat IgG. I
always found that you need a relatively high pH (close to 9) to really
make a difference. You would get closer to that pH with Tris because of
it's pK value, Imidazol is lower (about 6.8 to 7.0 I believe).
So under the conditions you used, you're not only having an unfortunate
pH, but also the freezing, when it is done slowly, causes the IgG to
seperate from the water during ice crystallization. So you are actually
creating small volumes between the ice crystals with extremely high
concentrations of IgG, which is only favourable for the molecules to
interact and precipitate.

So I would recommend a higher pH, fast freezing, maybe in the presence
of 50% glycerol, which keeps the mix liquid down to temperatures of
about -50°C.

Why does serum not give the same problem? Maybe because of the high
concentration of serum albumin (which is around 5% or something).
Albumin, through its high content of hydrophobic aminoacids, associates
with hydrophobic molecules like rabbit IgG, rendering the surface of the
complex more hydrophilic. So they won't precipitate that easily anymore.
With your purified IgG you're looking at a population of similar
proteins, which makes it easier to get a precipitate.

Hope your hair gets restored again!


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