drm21 at mole.bio.cam.ac.uk
Wed Sep 3 09:36:42 EST 1997
In article <5ujkkc$apn$1 at elna.ethz.ch>, 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.
For your application, would it be possible to switch the buffer while the
protein is still bound to the purification matrix? The Ni/His6 interaction
doesn't actually require the 4M Urea, so you could gradually dilute it out,
or do an entirely native purificiation....
You could then either elute the protein with imidazole and cleave with
rTEV-protease, or possibly cleave the protein on the column. In the latter
case, you wouldn't need any imidazol, and your protein would be free of
contaminating His6-fragments (but not free of rTEV-protease).
Hope it works,
D.R.Micklem, Time flies like an arrow...
Wellcome/CRC Institute, Fruit flies like a banana.
Cambridge CB2 1QR, UK Email:drm21 at mole.bio.cam.ac.uk
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