Achim Recktenwald, PhD
ARecktenwald at StressGen.com
Tue Sep 12 17:00:16 EST 2000
> Hi. There are, IME, two buggers that cause 'difficult' aggregation (i.e
> changing away from the pI does not help - which of course you should
> try). The first is proteolytic degradation. The second is the presence
> of hydrophobic patches on the protein (this how degradation effects
> aggregation). You can test for the second possibility by keeping the
> prep at 4C, or adding up to about 33% glycerol or ethylene glycol.
> Both approaches should inhibit hydrophobic interactions.
Another option is to add or dialyze against up to 25% sucrose.
When dialyzing against sucrose you loose a lot of volume = concentrate the
protein. With 25% the concentration factor is about 2 - 3 fold, depending on
the dialysis membrane and buffers used. Since this concentration method is
very slow and gentle, it might help to get your protein beyond this magical
limit of 5 mg/mL - in addition, sucrose inhibits precipitation.
More information about the Proteins