djs17 at york.ac.uk wrote:
>> Hia,
> Has anyone had problems with centricon-10 columns aggregating proteins?
> We had a sample that sedimented at 3s before concentration, and 20 s
> afterwards. It is known to be a tetramer of around 44 kDa, so the larger
> sedimentation coefficient is due to aggregation.
>> your views please....
>> Dave.
Centricons can definitely cause aggregation and precipitation, through
at least two different mechanisms.
One mechanism is simply that the concentration right at the membrane
surface can get extremely high (much higher than the bulk
concentration). You can reduce this effect somewhat by slowing down the
rate of concentration, allowing more time for diffusion.
The second effect is due to surface binding and adsorption of your
protein, which leads to conformational changes that promote aggregation.
The culprit here is often the ultrafiltration membrane (and again the
high local concentration promotes the binding), but it can also be the
container walls. People have had some success in reducing this problem
by pre-treating the Centricon's with glycerol. This presumably covers
up some of the 'sticky' sites, but the drawback is that even after you
pour out the glycerol some will remain and some will be present in your
concentrated sample.
John Philo, Alliance Protein Laboratories
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