Re: [ccp4bb] pH gradient in Mono Q

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

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Subject: POST-DOCTORAL POSITION University of Nebraska, Lincoln, NE
From: Hideaki Moriyama hmoriyama2 {- at -} UNL {- dot -} EDU
Date: 2008-06-25

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Subject: Re: pH gradient in Mono Q
From: "Nadir T {- dot -} Mrabet" Nadir {- dot -} Mrabet {- at -} MEDECINE {- dot -} UHP-NANCY {- dot -} FR
Date: 2008-06-26

Subject: Re: pH gradient in Mono Q
From: Matthew Chu linghonmatthew {- at -} GMAIL {- dot -} COM
Date: 2008-06-26

Thank you for all your advices.

I usually employ salt gradient in MonoQ for my proteins, but I just wonder
if we can use pH gradient in the same column and see if it can improve the
separation.

I know the condition that my protein binds to Mono Q, so I will try to
titrate toward its pI to reduce the affinity of the column. I am going to
try Tris / phosphate buffer first, will let you guys know the results.

Really appreciate for all your advices again.

Matt

2008/6/25 John A. Newitt :

> At 1:50 PM -0400 6/24/08, R.M. Garavito wrote:
>
> Matthew,
>>
>> You're not going to ruin your column, but you won't get great performance
>> either. Elution by pH change is a very common method, but getting a really
>> linear pH gradient is very hard. The Mono Q matrix is a strong anion
>> exchanger, meaning that it is insensitive to pH changes, i.e., you can't
>> titrate it smoothly with acid or base. DEAE resins, which are weak anion
>> exchangers, have a nice pH titration curve and lend themselves better to
>> elution by pH change. This is the reason chromatofocusing is not a commonly
>> used method, and its expensive.
>>
>
> There is a company the sells a proprietary buffer system and gradient
> programming calculator to create a stable pH gradient for separation on a
> MonoQ column or other strong ion exchanger.
>
>
>
> My problem with pH gradient techniques is that they don't work very well
> unless your protein is happy in low ionic strength buffers, which is almost
> never the case with my projects. This company now claims that it can create
> the pH gradient with NaCl present, but I haven't tried this yet.
>
> - John
> --
>
>

--
----------------------------------------------------------------------------
Matthew LH Chu
PhD Student
School of Pharmacy and Pharmaceutical Sciences
University of Manchester
----------------------------------------------------------------------------

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

Previous message:
Subject: POST-DOCTORAL POSITION University of Nebraska, Lincoln, NE
From: Hideaki Moriyama hmoriyama2 {- at -} UNL {- dot -} EDU
Date: 2008-06-25

Next message:
Subject: Re: pH gradient in Mono Q
From: "Nadir T {- dot -} Mrabet" Nadir {- dot -} Mrabet {- at -} MEDECINE {- dot -} UHP-NANCY {- dot -} FR
Date: 2008-06-26