Hi Fred,
I doubt His-trap column be any better. I never found much a difference
between these resins or columns. If you want to try, use His-Trap HP, not
His-Trap FF.
Doing batch binding may help you figure out the problem. In some cases,
protein binds better in batch mode. You can take beads out at time intervals
up to overnight for binding analysis.
I guess not working means the protein in the flow-through. Sometimes for
native purification, proteins don't come out of a column. But never
experienced anything like this under denatured conditions.
Good luck,
Chun
-----Original Message-----
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Fred
Sent: Wednesday, January 28, 2009 12:08 PM
To: [log in to unmask]
Subject: Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind
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 /<[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 *
>>
>>
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