Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind

- Protein crystallography

Main steps:

- Protein purification
- Crystallisation

Special:

- Programs for crystallography
- X-ray detectors

Basic tutorials:

   - Chemistry
   - Protein
   - Peptide
   - Amino Acids

Xtal community:

- CCP4BB

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: sticky crystals
From: Jeff Speir speir {- at -} SCRIPPS {- dot -} EDU
Date: 2009-01-28

Next message:
Subject: Re: small lines in diffraction pattern
From: Jacob Keller j-keller2 {- at -} MD {- dot -} NORTHWESTERN {- dot -} EDU
Date: 2009-01-28

Subject: Re: [OFF TOPIC] his-tag doesn't bind
From: "Nadir T {- dot -} Mrabet" Nadir {- dot -} Mrabet {- at -} MEDECINE {- dot -} UHP-NANCY {- dot -} FR
Date: 2009-01-28

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: Nadir.Mrabet@medecine.uhp-nancy.fr

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 // schrieb am *Di, 27.1.2009:
>> *
>>
>> *Von: Fred
>> 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 *
>>
>>
>
>

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: sticky crystals
From: Jeff Speir speir {- at -} SCRIPPS {- dot -} EDU
Date: 2009-01-28

Next message:
Subject: Re: small lines in diffraction pattern
From: Jacob Keller j-keller2 {- at -} MD {- dot -} NORTHWESTERN {- dot -} EDU
Date: 2009-01-28