Re: [ccp4bb] Reducing Agent Tips?

- Protein crystallography

Main steps:

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

Previous message:
Subject: Reducing Agent Tips?
From: Jeremiah Farelli jfarelli {- at -} BU {- dot -} EDU
Date: 2009-10-05

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

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

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:

type="cite">

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: Reducing Agent Tips?
From: Jeremiah Farelli jfarelli {- at -} BU {- dot -} EDU
Date: 2009-10-05

Next message:
Subject: Re: Reducing Agent Tips?
From: Ezra Peisach epeisach {- at -} GMAIL {- dot -} COM
Date: 2009-10-05