Problem with concentrating His Tag protein

D.K. no.email at thanks.to.spam.net
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. 

Your choices: 

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.

DK


 there is  and some 
stabilizers



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