Re: [ccp4bb] Reducing Agent Tips?

- Protein crystallography

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

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Subject: Re: Reducing Agent Tips?
From: Roger Rowlett rrowlett {- at -} COLGATE {- dot -} EDU
Date: 2009-10-05

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From: Pavel Afonine PAfonine {- at -} LBL {- dot -} GOV
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Subject: Re: Reducing Agent Tips?
From: Ezra Peisach epeisach {- at -} GMAIL {- dot -} COM
Date: 2009-10-05

Have you considered seeing if anyone has an anaerobic chamber you can
work in?

Ezra

On 10/05/2009 11:39 AM, Roger Rowlett wrote:
> Of these, typically TCEP>DTT>BME in terms of effectiveness in
> maintaining reduced Cys groups. Some proteins, when purified, require
> obscenely large reducing agent concentrations to keep them stable. One
> of our "horror show" proteins required 100 mM DTT + 10 uM EDTA to
> remain stable. You should consider including EDTA in the solution to
> sequester metal ions (if this is compatible with your protein). Metal
> ions, including the ubiquitious trace Zn2+, efficiently catalyze the
> oxidation of sulfhydryls. Include 1 uM or more if your protein will
> tolerate it.
>
> Cheers.
>
> Jeremiah Farelli wrote:
>> Hello all,
>>
>> Anyone have any tips for reducing agents for use with the following
>> crystallization conditions:
>>
>> 100 mM hepes, pH 7.5, 24% PEG 1500
>>
>> or
>>
>> 100 mM Tris, pH 8.0, 24% PEG 1500
>>
>> I've tried BME and DTT (1 mM). Currently trying out TCEP. The protein
>> seems to be sensitive to oxidation, with a selenomet-derivative even more so.
>>
>> Thanks!
>>
> --
> ------------------------------------------------------------------------
> Roger S. Rowlett
> Professor
> Department of Chemistry
> Colgate University
> 13 Oak Drive
> Hamilton, NY 13346
>
> tel: (315)-228-7245
> ofc: (315)-228-7395
> fax: (315)-228-7935
> email: rrowlett@colgate.edu

CCP4bb navigation

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

Previous message:
Subject: Re: Reducing Agent Tips?
From: Roger Rowlett rrowlett {- at -} COLGATE {- dot -} EDU
Date: 2009-10-05

Next message:
Subject: Re: anisotropic data
From: Pavel Afonine PAfonine {- at -} LBL {- dot -} GOV
Date: 2009-10-05