yona at alomone.com
Sun Mar 24 06:28:19 EST 2002
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.
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