Two things to be mentioned.
* IDA columns bear an overall negative charge. I expect this behavior
holds true with NTA gels. Hence salt, (>= 0.5 M NaCl) must be present in
your adsoprtion buffer to quench possible repulsive electrostatic
interactions.
* You are dealing with protein adsoption by coordination bond formation
to a metal-chelate. Coordination bond lentghs decrease (and binding
improves) as ionic strength increases, so a 1-2 M salt concentration in
you buffer may turn out to be appropriate.
HTH,
Nadir Mrabet
--
Pr. Nadir T. Mrabet
Cellular & Molecular Biochemistry
INSERM U-724
Nancy University, School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax: +33 (0)3.83.68.32.79
E-mail: [log in to unmask]
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 /<[log in to unmask]>/ schrieb am *Di, 27.1.2009:
>> *
>>
>> *Von: Fred <[log in to unmask]>
>> Betreff: [ccp4bb] [OFF TOPIC] his-tag doesn't bind
>> An: [log in to unmask]
>> 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 *
>>
>>
>
>
