yuyuan at students.wisc.edu
Sat Mar 30 17:08:05 EST 2002
IgG is protein. Please check your buffer to see if the concentration is too
high. If so, the protein will aggregate.
yona bismuth <yona at alomone.com> wrote in message
news:9cce8819.0203240328.4f75be9 at posting.google.com...
> I am purifiying polyclonal antibodies from rabbit serum with protein A
> and I have some problems of IgG aggregates: I elute IgG from the
> column with 20mM Sodium Citrate pH 3 (the IgG is coming out between
> pH6.4 to pH 4.6) and immediately titrate them with TRIS 1M pH8 (to get
> a final concentration of 100mM TRIS). The pH before and after the PBS
> dialysis is around 7.4.
> After one day at 4 degres Celsius the IgG solution begins to forms
> some aggregates and after filtration, the concentration of IgG is much
> lower: I lost most part of my antibodies.
> I have found that when the concentration is lower than 1mg/ml it does
> not aggregate and also that the aggregation occurs at 4 degres Celsius
> and not when the temperature is higher. I want to keep the temperature
> at 4 degres Celsius (a higher temperature can lead to degradation of
> the IgG) and have a concentration of IgG higher than 1mg/ml.
> The pH of the IgG-PBS solution is different from the pI of the IgG (I
> am still checking this point ) so I do not think the reason of the
> aggregates formation is the pH.
> These aggregates are concentration and temperature dependend and it
> could be that there are cryoglobulins. I have found that it is
> possible to recover them from the aggregate by warming the solution
> but they can lose their activity.
> I just want to avoid the aggregates to form.
> If someone know about this problem and could help me it could be nice.
> thank you
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