Just to let you know. No way, Talon also don't work. I am gonna try the
GE His-trap column.
Fred wrote:
> 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 *
>>
>>
