Problem with concentrating His Tag protein
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Tue Apr 29 12:34:03 EST 2003
In article <20030428234731.19647.qmail at ww02.hostica.com>, jfritz at scripps.edu wrote:
>I have a 6X His Tag protein that is secreted from 293 EBNA cells and is
> purified nicely using an antibody specific for the protein we work with that
> is fused to the His tag. We elute from the column using DEA pH 11.5 and
> neutralize with Sodium Phosphate pH 5.6
>The problem we are having is concentrating the protein using various
> concentrators from Millipore and Centricon. I lose over half of the protein
> after using the concentrator and it appears the protein precipitates out of
> solution while concentrating. Has anyone had this problem and if so I would
> really appreciate any suggestions and hints he/she can provide.
Very common thing. It is because there is a gradient of
protein concentration near membrane and right next to it
it can get very high. Meny proteins have limited solubility,
so they precipitate when the threashold is exceeded.
1. Find a "better" buffer that helps to increase your protein
solubility (choice of pH and ionic composition; sometimes
0.1% of mild detegent is acceptable but keep in mind that
detegent will get concentrated too).
2. Concentrate without untrafiltration (is ammonium sulphate
precipitation an option?).
3. Vortex Centricon every ~ 10 min between spins. This minimizes
the layer near membrane where protein is at too high concentration.
there is and some
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