krasel at wpxx02.toxi.uni-wuerzburg.de
Wed Sep 3 08:10:03 EST 1997
Patricia Werner (werner at inw.agrl.ethz.ch) 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.
Sounds like the protein is precipitating on the membrane. Maybe you could
try dialysis instead.
Can't you exchange the buffer on the column? I.e. eluting in Imidazol
without the urea?
/* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */
/* D-97078 Wuerzburg, Germany email: phak004 at rzbox.uni-wuerzburg.de SP3 */
/* "Science is the game we play with God to find out what His rules are." */
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