Hi everyone,
Thanks for answer my question. Just to add some more notes regarding to
my expression system. The insert-vector (pET28) has been sequenced and
the his-tag is N-terminal. The anti his-tag WB is positive and the
binding buffer's pH is 8.2 (double-checked).
I had already experienced the same problem before, which I solved just
increasing urea from 6M to 8M. Now, I have reached GndHCl 6M and no
binding at all.
I'm currently running a SDS-PAGE with samples eluted from Talon and let
you know the results.
All the Best,
Fred
--- Fred /<ccp4bb.l...@gmail.com>/ schrieb am *Di, 27.1.2009:
*
*Von: Fred <ccp4bb.l...@gmail.com>
Betreff: [ccp4bb] [OFF TOPIC] his-tag doesn't bind
An: CCP4BB@JISCMAIL.AC.UK
Datum: Dienstag, 27. Januar 2009, 22:00
*
*Hi ccp4 list,
I am trying to purify a his-tag protein by metal affinity chromatography.
The
protein was expressed in inclusion bodies and its his-tag doesn't bind the
Qiagen Ni resin in denatured conditions (urea 8M or GndHCl 6M). Playing with
NaCl and detergents didn't help much.
Any help is appreciated.
Fred *
