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

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

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Subject: Re: OT: VectorNTI alternatives - 4 Mac?
From: Jovine Luca luca {- dot -} jovine {- at -} KI {- dot -} SE
Date: 2009-01-28

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Subject: Re: OT: VectorNTI alternatives - 4 Mac?
From: Peter Keller pkeller {- at -} GLOBALPHASING {- dot -} COM
Date: 2009-01-28

Subject: Re: [OFF TOPIC] his-tag doesn't bind
From: Fred ccp4bb {- dot -} list {- at -} GMAIL {- dot -} COM
Date: 2009-01-28

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 *
>
>

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

Previous message:
Subject: Re: OT: VectorNTI alternatives - 4 Mac?
From: Jovine Luca luca {- dot -} jovine {- at -} KI {- dot -} SE
Date: 2009-01-28

Next message:
Subject: Re: OT: VectorNTI alternatives - 4 Mac?
From: Peter Keller pkeller {- at -} GLOBALPHASING {- dot -} COM
Date: 2009-01-28