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: [email protected]
