centricon concentrators

Julie jroberts at rascal.med.harvard.edu
Thu Sep 4 08:09:05 EST 1997


I have been using the Pharmacia PD-10 columns to desalt my his-bind
elute.  I equilibrate the columns with 25 ml 20 mM Tris 8 then load the
column with my sample (2.5 ml) then eulte the sample with 3.5 ml 20 mM
Tris 8. I have good recovery with this.

Julie Roberts
jroberts at rascal.med.harvard.edu



Patricia Werner wrote:

> I hope somebody can help me! I am isolating a protein with His-Tag.
> In order to get rid of the poly-histidine I am doing a rTEV-protease
> digestion. The problem is, that in the end of the isolation the
> protein
> is in 4M Urea, 500 mM Imidazol, but the protein doesn't work with such
>
> high Urea concentrations.
>
> So I am trying to change the buffer by centrifugation with centricon
> concentrators from amicon. But yield is only about 10%. The protein
> seems
> to dissapear during the concentration procedure. I can find it neither
>
> in the flow-through, nor in the retentate.
>
> Thanks in advance
>
> Please send e-mail to: werner at inw.agrl.ethz.ch






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